Xylanases, Nucleic Acids Encoding Them and Methods for Making and Using Them

ABSTRACT

The invention relates to enzymes having xylanase, mannanase and/or glucanase activity, e.g., catalyzing hydrolysis of internal β-1,4-xylosidic linkages or endo-β-1,4-glucanase linkages; and/or degrading a linear polysaccharide beta-1,4-xylan into xylose. Thus, the invention provides methods and processes for breaking down hemicellulose, which is a major component of the cell wall of plants, including methods and processes for hydrolyzing hemicelluloses in any plant or wood or wood product, wood waste, paper pulp, paper product or paper waste or byproduct. In addition, methods of designing new xylanases, mannanases and/or glucanases and methods of use thereof are also provided. The xylanases, mannanases and/or glucanases have increased activity and stability at increased pH and temperature.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 12/681,604, filed on 14 Sep. 2010; which is the national phaseapplication claiming the benefit of priority under 35 U.S.C. §371 toPatent Cooperation Treaty (PCT) Application No. PCT/US2008/072030 havingan international filing date of 1 Aug. 2008 (published as WO2009/045627, on 9 Apr. 2009); which claims the benefit of priority under35 U.S.C. §119(e) to U.S. Provisional Application No. 60/977,348 filed 3Oct. 2007. The contents of the above applications are incorporated byreference herein in their entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made in part with Government support under ContractNo. DOE 1435-04-03-CA-70224, awarded by the Department of Energy. TheGovernment may have certain rights in the invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application was filed electronically via the USPTO EFS-WEB server,as authorized and set forth in MPEP §1730 II.B.2.(a)(A), and thiselectronic filing includes an electronically submitted sequence (SEQ ID)listing; the entire content of this sequence listing is hereinincorporated by reference for all purposes.

FIELD OF THE INVENTION

This invention relates generally to enzymes, polynucleotides encodingthe enzymes, the use of such polynucleotides and polypeptides and morespecifically to enzymes having xylanase activity, e.g., endoxylanaseactivity, and/or catalyzing hydrolysis of internal β-1,4-xylosidiclinkages or endo-β-1,4-glucanase linkages; and/or degrading a linearpolysaccharide beta-1,4-xylan into xylose; or, a glucanase activity,e.g., an endoglucanase activity, for example, catalyzing hydrolysis ofinternal endo-β-1,4- and/or 1,3-glucanase linkages, a xylanase activity,and/or a mannanase activity. Thus, the invention provides methods andprocesses for breaking down hemicellulose, which is a major component ofthe cell wall of plants, including methods and processes for hydrolyzinghemicelluloses in any organic compound, plant or wood or wood product orbyproduct, wood waste, paper pulp, paper product or paper waste orbyproduct. The invention further provides methods and processes forbreaking down plant matter containing cellulose and/or hemicelluloseinto simple sugars using the “cocktails” of the invention.

BACKGROUND

Xylanases (e.g., endo-1,4-beta-xylanase, EC 3.2.1.8) hydrolyze internalβ-1,4-xylosidic linkages in xylan to produce smaller molecular weightxylose and xylo-oligomers. Xylans are polysaccharides formed from1,4-β-glycoside-linked D-xylopyranoses. Xylanases are of considerablecommercial value, being used in the food industry, for baking and fruitand vegetable processing, breakdown of agricultural waste, in themanufacture of animal feed and in pulp and paper production. Xylanasesare formed by fungi and bacteria.

Arabinoxylans are major non-starch polysaccharides of cerealsrepresenting 2.5-7.1% w/w depending on variety and growth conditions.The physicochemical properties of this polysaccharide are such that itgives rise to viscous solutions or even gels under oxidative conditions.In addition, arabinoxylans have high water-binding capacity and may havea role in protein foam stability. All of these characteristics presentproblems for several industries including brewing, baking, animalnutrition and paper manufacturing. In brewing applications, the presenceof xylan results in wort filterability and haze formation issues. Inbaking applications (especially for cookies and crackers), thesearabinoxylans create sticky doughs that are difficult to machine andreduce biscuit size. In addition, this carbohydrate is implicated inrapid rehydration of the baked product resulting in loss of crispinessand reduced shelf-life. For monogastric animal feed applications withcereal diets, arabinoxylan is a major contributing factor to viscosityof gut contents and thereby adversely affects the digestibility of thefeed and animal growth rate. For ruminant animals, these polysaccharidesrepresent substantial components of fiber intake and more completedigestion of arabinoxylans would facilitate higher feed conversionefficiencies.

There remains a need in the art for xylanases to be used in the paperand pulp industry, for example, where the enzyme is active in thetemperature range of 65° C. to 75° C. and at a pH of approximately 10.Additionally, an enzyme useful in the paper and pulp industry woulddecrease the need for bleaching chemicals, such as chlorine dioxide.

Additionally, there remains a need to provide efficient, low costprocesses and compositions for producing bioalcohols, biofuels and/orbiofuel-(e.g., bioethanol-, propanol-, butanol- and/or methanol-) byconversion of biomass. An enzyme or enzyme “cocktail” could provide aroute to convert biomass into sugars that could then be fermented intobiofuels.

SUMMARY OF THE INVENTION

The invention provides enzymes having: xylanase activity, e.g.,endoxylanase activity, and/or catalyzing hydrolysis of internalβ-1,4-xylosidic linkages or endo-β-1,4-glucanase linkages; and/or,having a glucanase activity, e.g., an endoglucanase activity, forexample, catalyzing hydrolysis of internal endo-β-1,4- and/or1,3-glucanase linkages, a xylanase activity, and/or a mannanaseactivity; and, nucleic acids encoding them, vectors and cells comprisingthem, probes for amplifying and identifying these xylanase-encodingnucleic acids, and methods for making and using these polypeptides andpeptides.

For example, the invention provides enzymes having xylanase (e.g.,endoxylanase activity), and compositions and methods comprising them,for hydrolyzing internal β-1,4-xylosidic linkages orendo-β-1,4-glucanase linkages, or hemicelluloses, in a wood, woodproduct, paper pulp, paper product or paper waste. In one aspect, thexylanase activity comprises catalyzing hydrolysis of xylan, e.g.,degrading a linear polysaccharide beta-1,4-xylan into a xylose. Thus,the invention provides methods and processes for breaking down axylan-comprising composition and/or a hemicellulose, which is a majorcomponent of the cell wall of plants.

In one aspect, the glucanase activity of a polypeptide or peptide of theinvention (which includes a protein or peptide encoded by a nucleic acidof the invention) comprises an endoglucanase activity, e.g., endo-1,4-and/or 1,3-beta-D-glucan 4-glucano hydrolase activity. In one aspect,the endoglucanase activity comprises catalyzing hydrolysis of1,4-beta-D-glycosidic linkages. In one aspect, the glucanase, e.g.,endoglucanase, activity comprises an endo-1,4- and/or1,3-beta-endoglucanase activity or endo-β-1,4-glucanase activity. In oneaspect, the glucanase activity (e.g., endo-1,4-beta-D-glucan 4-glucanohydrolase activity) comprises hydrolysis of 1,4-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (e.g., carboxy methylcellulose and hydroxy ethyl cellulose) lichenin, beta-1,4 bonds in mixedbeta-1,3 glucans, such as cereal beta-D-glucans and other plant materialcontaining cellulosic parts. In one aspect, the glucanase, xylanase, ormannanase activity comprises hydrolyzing a glucan or otherpolysaccharide to produce a smaller molecular weight polysaccharide oroligomer. In one aspect, the glucan comprises a beta-glucan, such as awater soluble beta-glucan.

The invention provides enzymes, compositions, methods and processes forhydrolyzing hemicelluloses in any organic matter, including cells,plants and/or wood or wood products, wood waste, paper pulp, paperproducts or paper waste or byproducts. The invention further providesmethods and processes for breaking down plant matter containingcellulose and/or hemicellulose into simple sugars using the “cocktails”of the invention.

In another aspect, the invention provides polypeptides havinglignocellulolytic (lignocellulosic) activity, e.g., a ligninolytic andcellulolytic activity, including, e.g., having a hydrolase activity,e.g., a glycosyl hydrolase activity, including cellulase, glucanase,xylanase, and/or mannanase activity, and nucleic acids encoding them,and methods for making and using them. The invention provides enzymesfor the bioconversion of any biomass, e.g., a lignocellulosic residue,into fermentable sugars or polysaccharides; and these sugars orpolysaccharides can be used as a chemical feedstock for the productionof alcohols such as ethanol, propanol, butanol and/or methanol,production of fuels, e.g., biofuels such as synthetic liquids or gases,such as syngas, and the production of other fermentation products, e.g.succinic acid, lactic acid, or acetic acid. Enzymes of the invention canbe added to bioconversion and other industrial processes continuously,in batches or by fed-batch methods. In another aspect, enzymes of theinvention can be recycled in bioconversion and other industrialprocesses, thereby lowering enzyme requirements.

In one aspect, the enzymes of the invention have an increased catalyticrate to improve the process of substrate (e.g., a lignocellulosicresidue, cellulose, bagasse) hydrolysis. This increased efficiency incatalytic rate leads to an increased efficiency in producing sugars orpolysaccharides, which can be useful in industrial, agricultural ormedical applications, e.g., to make a biofuel or an alcohol such asethanol, propanol, butanol and/or methanol. In one aspect, sugarsproduced by hydrolysis using enzymes of this invention can be used bymicroorganisms for alcohol (e.g., ethanol, propanol, butanol and/ormethanol) production and/or fuel (e.g., biofuel) production.Additionally, the sugars produced by hydrolysis using the enzymes of theinvention can be used by microorganisms for the production of otherfermentation products, e.g. succinic acid, lactic acid, or acetic acid.

The invention provides industrial, agricultural or medical applications:e.g., biomass to biofuel, e.g., ethanol, propanol, butanol and/ormethanol, using enzymes of the invention having decreased enzyme costs,e.g., decreased costs in biomass to biofuel conversion processes. Thus,the invention provides efficient processes for producing bioalcohols,biofuels and/or biofuel—(e.g., bioethanol-, propanol-, butanol- and/ormethanol-) comprising compositions, including synthetic, liquid or gasfuels comprising a bioalcohol, from any biomass.

In one aspect, enzymes of the invention, including the enzyme“cocktails” of the invention (“cocktails” meaning mixtures of enzymescomprising at least one enzyme of this invention), are used to hydrolyzethe major components of a lignocellulosic biomass, or any compositioncomprising cellulose and/or hemicellulose (lignocellulosic biomass alsocomprises lignin), e.g., seeds, grains, tubers, plant waste (such as ahay or straw, e.g., a rice straw or a wheat straw, or any the dry stalkof any cereal plant) or byproducts of food processing or industrialprocessing (e.g., stalks), corn (including cobs, stover, and the like),grasses (e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g., Panicum species, such as Panicum virgatum), wood (includingwood chips, processing waste, such as wood waste), paper, pulp, recycledpaper (e.g., newspaper); also including a monocot or a dicot, or amonocot corn, sugarcane or parts thereof (e.g., cane tops), rice, wheat,barley, switchgrass or Miscanthus; or a dicot oilseed crop, soy, canola,rapeseed, flax, cotton, palm oil, sugar beet, peanut, tree, poplar orlupine; or, woods or wood processing byproducts, such as wood waste,e.g., in the wood processing, pulp and/or paper industry, in textilemanufacture and in household and industrial cleaning agents, and/or inbiomass waste processing.

In one aspect, enzymes of the invention are used to hydrolyze cellulosecomprising a linear chain of β-1,4-linked glucose moieties, and/orhemicellulose as a complex structure that varies from plant to plant. Inone aspect, enzymes of the invention are used to hydrolyzehemicelluloses containing a backbone of β-1,4 linked xylose moleculeswith intermittent branches of arabinose, galactose, glucuronic acidand/or mannose. In one aspect, enzymes of the invention are used tohydrolyze hemicellulose containing non-carbohydrate constituents such asacetyl groups on xylose and ferulic acid esters on arabinose. In oneaspect, enzymes of the invention are used to hydrolyze hemicellulosescovalently linked to lignin and/or coupled to other hemicellulosestrands via diferulate crosslinks.

In one aspect, the compositions and methods of the invention are used inthe enzymatic digestion of biomass and can comprise use of manydifferent enzymes, including the cellulases and hemicellulases.Lignocellulosic enzymes used to practice the invention can digestcellulose to monomeric sugars, including glucose. In one aspect,compositions used to practice the invention can include mixtures ofenzymes, e.g., glycosyl hydrolases, glucose oxidases, xylanases,xylosidases (e.g., β-xylosidases), cellobiohydrolases, and/orarabinofuranosidases or other enzymes that can digest hemicellulose tomonomer sugars. Mixtures of the invention can comprise, or consist of,only enzymes of this invention, or can include at least one enzyme ofthis invention and another enzyme, which can also be a lignocellulosicenzyme and/or any other enzyme.

In one aspect, the enzymes of the invention have a glucanase, e.g., anendoglucanase, activity, e.g., catalyzing hydrolysis of internalendo-β-1,4- and/or β-1,3-glucanase linkages. In one aspect, theendoglucanase activity (e.g., endo-1,4-beta-D-glucan 4-glucano hydrolaseactivity) comprises hydrolysis of 1,4- and/or β-1,3-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (e.g., carboxy methylcellulose and hydroxy ethyl cellulose) lichenin, beta-1,4 bonds in mixedbeta-1,3 glucans, such as cereal beta-D-glucans or xyloglucans and otherplant material containing cellulosic parts.

In alternative embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having at least one conservative aminoacid substitution and retaining its xylanase, a mannanase and/or aglucanase activity; or, wherein the at least one conservative amino acidsubstitution comprises substituting an amino acid with another aminoacid of like characteristics; or, a conservative substitution comprises:replacement of an aliphatic amino acid with another aliphatic aminoacid; replacement of a Serine with a Threonine or vice versa;replacement of an acidic residue with another acidic residue;replacement of a residue bearing an amide group with another residuebearing an amide group; exchange of a basic residue with another basicresidue; or replacement of an aromatic residue with another aromaticresidue;

In alternative embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having a xylanase (e.g., anendoxylanase), a mannanase and/or a glucanase activity but lacking asignal sequence, a prepro domain, a dockerin domain, and/or acarbohydrate binding module (CBM); and in one aspect, the carbohydratebinding module (CBM) comprises, or consists of, a xylan binding module,a cellulose binding module, a lignin binding module, a xylose bindingmodule, a mannanse binding module, a xyloglucan-specific module and/or aarabinofuranosidase binding module.

In alternative embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having a xylanase (e.g., anendoxylanase), a mannanase and/or a glucanase activity furthercomprising a heterologous sequence; and in one aspect, the heterologoussequence comprises, or consists of a sequence encoding: (i) aheterologous signal sequence, a heterologous carbohydrate bindingmodule, a heterologous dockerin domain, a heterologous catalytic domain(CD), or a combination thereof; (ii) the sequence of (ii), wherein theheterologous signal sequence, carbohydrate binding module or catalyticdomain (CD) is derived from a heterologous enzyme; or, (iii) a tag, anepitope, a targeting peptide, a cleavable sequence, a detectable moietyor an enzyme; and in one aspect, the heterologous carbohydrate bindingmodule (CBM) comprises, or consists of, a xylan binding module, acellulose binding module, a lignin binding module, a xylose bindingmodule, a mannanse binding module, a xyloglucan-specific module and/or aarabinofuranosidase binding module; and in one aspect, the heterologoussignal sequence targets the encoded protein to a vacuole, theendoplasmic reticulum, a chloroplast or a starch granule.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising (a) a nucleic acid (polynucleotide) encoding at least onepolypeptide, wherein the nucleic acid comprises a sequence having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete(100%) sequence identity to:

(i) the nucleic acid (polynucleotide) sequence of SEQ ID NO:1 having oneor more nucleotide residue changes (or the equivalent thereof) as setforth in Table 1, or having at least one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen or eighteen, or some or all of the followingnucleotide residue changes: the codon encoding amino acid residue 4changed from ACC to AAC; the codon encoding amino acid residue 4 changedfrom ACC to CGC; the codon encoding amino acid residue 4 changed fromACC to CAC; the codon encoding amino acid residue 9 changed from CCC toGAC; the codon encoding amino acid residue 17 changed from TTC to GTC;the codon encoding amino acid residue 21 changed from TTC to TAC; thecodon encoding amino acid residue 33 changed from CTG to GCG; the codonencoding amino acid residue 38 changed from CGT to CAC; the codonencoding amino acid residue 44 changed from AGC to ACG; the codonencoding amino acid residue 63 changed from ATC to GTC; the codonencoding amino acid residue 73 changed from GGC to TAC; the codonencoding amino acid residue 73 changed from GGC to GAG; the codonencoding amino acid residue 73 changed from GGC to GTC; the codonencoding amino acid residue 108 changed from TTC to AAG; the codonencoding amino acid residue 125 changed from CAG to TAC; the codonencoding amino acid residue 150 changed from GTA to GCC; the codonencoding amino acid residue 188 changed from AGC to GAG; and/or, thecodon encoding amino acid residue 189 changed from TCC to CAG; or

(ii) the nucleic acid (polynucleotide) sequence of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21 or SEQ ID NO:23;

wherein the nucleic acid of (i) or (ii) encodes at least one polypeptidehaving a xylanase, a mannanase and/or a glucanase activity, or encodes apolypeptide or peptide capable of generating a xylanase, a mannanaseand/or a glucanase specific antibody (a polypeptide or peptide that actsas an epitope or immunogen),

(b) the nucleic acid (polynucleotide) of (a), wherein the sequenceidentities are determined: (A) by analysis with a sequence comparisonalgorithm or by a visual inspection, or (B) over a region of at leastabout 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 ormore residues, or over the full length of a cDNA, transcript (mRNA) orgene;

(c) the nucleic acid (polynucleotide) of (a) or (b), wherein thesequence comparison algorithm is a BLAST version 2.2.2 algorithm where afiltering setting is set to blastall -p blastp -d “nr pataa”-F F, andall other options are set to default;

(d) a nucleic acid (polynucleotide) encoding at least one polypeptide orpeptide, wherein the nucleic acid comprises a sequence that hybridizesunder stringent conditions to a nucleic acid comprising the nucleic acid(polynucleotide) sequence of SEQ ID NO:1 having one or more nucleotideresidue changes (or the equivalent thereof) as set forth in Table 1, orhaving at least one, two, three, four, five, six, seven, eight, nine,ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen oreighteen, or some or all of the following nucleotide residue changes:the codon encoding amino acid residue 4 changed from ACC to AAC; thecodon encoding amino acid residue 4 changed from ACC to CGC; the codonencoding amino acid residue 4 changed from ACC to CAC; the codonencoding amino acid residue 9 changed from CCC to GAC; the codonencoding amino acid residue 17 changed from TTC to GTC; the codonencoding amino acid residue 21 changed from TTC to TAC; the codonencoding amino acid residue 33 changed from CTG to GCG; the codonencoding amino acid residue 38 changed from CGT to CAC; the codonencoding amino acid residue 44 changed from AGC to ACG; the codonencoding amino acid residue 63 changed from ATC to GTC; the codonencoding amino acid residue 73 changed from GGC to TAC; the codonencoding amino acid residue 73 changed from GGC to GAG; the codonencoding amino acid residue 73 changed from GGC to GTC; the codonencoding amino acid residue 108 changed from TTC to AAG; the codonencoding amino acid residue 125 changed from CAG to TAC; the codonencoding amino acid residue 150 changed from GTA to GCC; the codonencoding amino acid residue 188 changed from AGC to GAG; and/or, thecodon encoding amino acid residue 189 changed from TCC to CAG,

wherein the polypeptide or peptide has a xylanase, a mannanase and/or aglucanase activity or is capable of generating a xylanase, a mannanaseand/or a glucanase specific antibody (a polypeptide or peptide that actsas an epitope or immunogen),

and the stringent conditions comprise a wash step comprising a wash in0.2×SSC at a temperature of about 65° C. for about 15 minutes;

(e) a nucleic acid (polynucleotide) encoding at least one polypeptide orpeptide, wherein the nucleic acid comprises a sequence that hybridizesunder stringent conditions to a nucleic acid comprising the sequence ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21 or SEQ ID NO:23,

and the stringent conditions comprise a wash step comprising a wash in0.2×SSC at a temperature of about 65° C. for about 15 minutes;

(f) the nucleic acid (polynucleotide) of any of (a) to (d) having alength of at least about 20, 25, 30, 50, 75, 100, 125, 150, 175, 200,225, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150 or more nucleotide residues, or the fulllength of a gene or a transcript;

(g) a nucleic acid (polynucleotide) encoding at least one polypeptidehaving a xylanase, a mannanase and/or a glucanase activity, wherein thepolypeptide comprises the sequence of SEQ ID NO:2, or enzymaticallyactive fragments thereof, has at least one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen or eighteen or some or all of the following aminoacid residue changes:

(h) a nucleic acid (polynucleotide) encoding at least one polypeptidehaving a xylanase, a mannanase and/or a glucanase activity, wherein thepolypeptide comprises the sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, orenzymatically active fragments thereof;

(i) (A) the nucleic acid (polynucleotide) of any of (a) to (h) andencoding a polypeptide having at least one conservative amino acidsubstitution and retaining its xylanase, a mannanase and/or a glucanaseactivity; or, (B) the nucleic acid of (i)(A), wherein the at least oneconservative amino acid substitution comprises substituting an aminoacid with another amino acid of like characteristics; or, a conservativesubstitution comprises: replacement of an aliphatic amino acid withanother aliphatic amino acid; replacement of a Serine with a Threonineor vice versa; replacement of an acidic residue with another acidicresidue; replacement of a residue bearing an amide group with anotherresidue bearing an amide group; exchange of a basic residue with anotherbasic residue; or replacement of an aromatic residue with anotheraromatic residue;

(j) the nucleic acid (polynucleotide) of any of (a) to (i) encoding apolypeptide having a xylanase, a mannanase and/or a glucanase activitybut lacking a signal sequence, a prepro domain, a dockerin domain,and/or a carbohydrate binding module (CBM);

(k) the nucleic acid (polynucleotide) of (j), wherein the carbohydratebinding module (CBM) comprises, or consists of, a xylan binding module,a cellulose binding module, a lignin binding module, a xylose bindingmodule, a mannanse binding module, a xyloglucan-specific module and/or aarabinofuranosidase binding module;

(1) the nucleic acid (polynucleotide) of any of (a) to (k) encoding apolypeptide having a xylanase, a mannanase and/or a glucanase activityfurther comprising a heterologous sequence;

(m) the nucleic acid (polynucleotide) of (1), wherein the heterologoussequence comprises, or consists of a sequence encoding: (A) aheterologous signal sequence, a heterologous carbohydrate bindingmodule, a heterologous dockerin domain, a heterologous catalytic domain(CD), or a combination thereof; (B) the sequence of (1), wherein theheterologous signal sequence, carbohydrate binding module or catalyticdomain (CD) is derived from a heterologous enzyme; or, (C) a tag, anepitope, a targeting peptide, a cleavable sequence, a detectable moietyor an enzyme;

(n) the nucleic acid (polynucleotide) of (1), wherein the heterologouscarbohydrate binding module (CBM) comprises, or consists of, a xylanbinding module, a cellulose binding module, a lignin binding module, axylose binding module, a mannanse binding module, a xyloglucan-specificmodule and/or a arabinofuranosidase binding module;

(o) the nucleic acid (polynucleotide) of (1), wherein the heterologoussignal sequence targets the encoded protein to a vacuole, theendoplasmic reticulum, a chloroplast or a starch granule; or

(p) a nucleic acid sequence (polynucleotide) fully (completely)complementary to the sequence of any of (a) to (o).

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid encoding at least one polypeptide having axylanase (e.g., an endoxylanase), a mannanase and/or a glucanaseactivity, wherein the polypeptide has a sequence as set forth in SEQ IDNO:2 having one or more changes as described herein and in Table 1, orenzymatically active fragments thereof, including the sequencesdescribed herein and in Table 1, and the Sequence Listing (all of thesesequences are “exemplary enzymes/polypeptides of the invention”), andenzymatically active subsequences (fragments) thereof and/orimmunologically active subsequences thereof (such as epitopes orimmunogens) (all “peptides of the invention”) and variants thereof (allof these sequences encompassing polypeptide and peptide sequences of theinvention) (or, hereinafter referred to as the exemplary polypeptidesequences of the inventions).

The invention provides isolated, synthetic or recombinant nucleic acidscomprising sequences completely complementary to all of these nucleicacid sequences of the invention (complementary (non-coding) and codingsequences also hereinafter collectively referred to as nucleic acidsequences of the invention).

In one aspect, the sequence identity is at least about 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% (complete) sequence identity (homology). Inone aspect, the sequence identity is over a region of at least about150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues,or the full length of a gene or a transcript. For example, the inventionprovides isolated, synthetic or recombinant nucleic acids comprising anucleic acid sequence of SEQ ID NO:1 having one or more mutations asdescribed herein, e.g., as described in Table 1 (the exemplarypolynucleotide sequences of this invention). The invention providesisolated, synthetic or recombinant nucleic acids encoding a polypeptidecomprising a sequence of SEQ ID NO:2 having one or more amino acidchange as described herein, e.g., as set forth in Table 1 (the exemplarypolypeptide sequences of this invention), and enzymatically activefragments thereof.

The invention provides isolated, synthetic or recombinant nucleic acidsencoding a polypeptide having xylanase (e.g., an endoxylanase), amannanase and/or a glucanase activity, wherein the nucleic acid has atleast one sequence modification of an exemplary sequence of theinvention, or, any sequence of the invention.

The invention provides isolated, synthetic or recombinant nucleic acidsencoding a polypeptide having xylanase (e.g., an endoxylanase), amannanase and/or a glucanase activity, wherein the nucleic acid has atleast one sequence modification of an exemplary nucleic acid of theinvention, wherein in one aspect the modifications (changes) are setforth in Table 1.

In one aspect, the invention also provides enzyme-encoding nucleic acidswith a common novelty in that they encode a novel subset of xylanases,or a Glade, comprising the “X14 module” (J. Bacteriol. 2002 August;184(15): 4124-4133). In one aspect, the invention also providesenzyme-encoding nucleic acids with a common novelty in that they encodea novel subset of xylanases, or a Glade, comprising the “X14 module”.Thus, in one aspect, the invention provides a novel genus of xylanasescomprising xylanase members of SEQ ID NO:2 having one or more mutationsas described herein, e.g., in Table 1.

In one aspect (optionally), the isolated, synthetic or recombinantnucleic acids of the invention have a xylanase (e.g., an endoxylanase),a mannanase and/or a glucanase activity, e.g., wherein the xylanaseactivity comprises catalyzing hydrolysis of internal β-1,4-xylosidiclinkages; comprises an endo-1,4-beta-xylanase activity; compriseshydrolyzing a xylan or an arabinoxylan to produce a smaller molecularweight xylose and xylo-oligomer; comprises hydrolyzing a polysaccharidecomprising a 1,4-β-glycoside-linked D-xylopyranose; compriseshydrolyzing a cellulose or a hemicellulose; comprises hydrolyzing acellulose or a hemicellulose in a wood, wood product, paper pulp, paperproduct or paper waste; comprises catalyzing hydrolysis of a xylan or anarabinoxylan in a feed or a food product; or, comprises catalyzinghydrolysis of a xylan or an arabinoxylan in a microbial cell or a plantcell. In one aspect, the xylanase activity comprises hydrolyzingpolysaccharides comprising 1,4-β-glycoside-linked D-xylopyranoses orhydrolyzing hemicelluloses, e.g., hydrolyzing hemicelluloses in a wood,wood product, paper pulp, paper product or paper waste. In one aspect,the arabinoxylan is a cereal arabinoxylan, such as a wheat arabinoxylan.

In one aspect, the xylanase, a mannanase and/or a glucanase activitycomprises catalyzing hydrolysis of polysaccarides, e.g., mannans orxylans, in a feed or a food product, such as a cereal-based animal feed,a wort or a beer, a milk or a milk product, a fruit or a vegetable. Inone aspect, the xylanase, a mannanase and/or a glucanase activitycomprises catalyzing hydrolysis of polysaccarides, e.g., mannans orxylans, in a microbial cell or a plant cell.

In one aspect, the xylanase, a mannanase and/or a glucanase activity isthermostable, e.g., wherein the polypeptide retains a xylanase, amannanase and/or a glucanase activity under conditions comprising atemperature range from about −100° C. to about −80° C., about −80° C. toabout −40° C., about −40° C. to about −20° C., about −20° C. to about 0°C., about 0° C. to about 5° C., about 5° C. to about 15° C., about 15°C. to about 25° C., about 25° C. to about 37° C., about 37° C. to about45° C., about 45° C. to about 55° C., about 55° C. to about 70° C.,about 70° C. to about 75° C., about 75° C. to about 85° C., about 85° C.to about 90° C., about 90° C. to about 95° C., about 95° C. to about100° C., about 100° C. to about 105° C., about 105° C. to about 110° C.,about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99°C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107°C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115°C. or more. In some embodiments, the thermostable polypeptides accordingto the invention retains activity, e.g., a xylanase, a mannanase and/ora glucanase activity, at a temperature in the ranges described above, atabout pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0,about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5,about pH 8.0, about pH 8.5, about pH 9.0, about pH 9.5, about pH 10.0,about pH 10.5, about pH 11.0, about pH 11.5, about pH 12.0 or more.

In one aspect, the xylanase, a mannanase and/or a glucanase activity isthermotolerant, e.g., wherein the polypeptide retains a xylanase, amannanase and/or a glucanase activity after exposure to a temperature inthe range from about −100° C. to about −80° C., about −80° C. to about−40° C., about −40° C. to about −20° C., about −20° C. to about 0° C.,about 0° C. to about 5° C., about 5° C. to about 15° C., about 15° C. toabout 25° C., about 25° C. to about 37° C., about 37° C. to about 45°C., about 45° C. to about 55° C., about 55° C. to about 70° C., about70° C. to about 75° C., about 75° C. to about 85° C., about 85° C. toabout 90° C., about 90° C. to about 95° C., about 95° C. to about 100°C., about 100° C. to about 105° C., about 105° C. to about 110° C.,about 110° C. to about 120° C., or 95° C., 96° C., 97° C., 98° C., 99°C., 100° C., 101° C., 102° C., 103° C., 104° C., 105° C., 106° C., 107°C., 108° C., 109° C., 110° C., 111° C., 112° C., 113° C., 114° C., 115°C. or more. The thermotolerant polypeptides according to the inventioncan retain activity, e.g. a xylanase, a mannanase and/or a glucanaseactivity, after exposure to a temperature in the range from about −100°C. to about −80° C., about −80° C. to about −40° C., about −40° C. toabout −20° C., about −20° C. to about 0° C., about 0° C. to about 5° C.,about 5° C. to about 15° C., about 15° C. to about 25° C., about 25° C.to about 37° C., about 37° C. to about 45° C., about 45° C. to about 55°C., about 55° C. to about 70° C., about 70° C. to about 75° C., about75° C. to about 85° C., about 85° C. to about 90° C., about 90° C. toabout 95° C., about 95° C. to about 100° C., about 100° C. to about 105°C., about 105° C. to about 110° C., about 110° C. to about 120° C., or95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103°C., 104° C., 105° C., 106° C., 107° C., 108° C., 109° C., 110° C., 111°C., 112° C., 113° C., 114° C., 115° C. or more. In some embodiments, thethermotolerant polypeptides according to the invention retains activity,e.g. a xylanase, a mannanase and/or a glucanase activity, after exposureto a temperature in the ranges described above, at about pH 3.0, aboutpH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH8.5, about pH 9.0, about pH 9.5, about pH 10.0, about pH 10.5, about pH11.0, about pH 11.5, about pH 12.0 or more.

In one aspect, the xylanase, a mannanase and/or a glucanase activity ofpolypeptides encoded by nucleic acids of the invention retain activityunder acidic conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH4.5, pH 4.0, pH 3.5, pH 3.0 or less (more acidic) pH, or, retain axylanase, a mannanase and/or a glucanase activity after exposure toacidic conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5,pH 4.0, pH 3.5, pH 3.0 or less (more acidic) pH; or, retain activityunder basic conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (morebasic) or, retain a xylanase, a mannanase and/or a glucanase activityafter exposure to basic conditions comprising about pH 7, pH 7.5 pH 8.0,pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 ormore (more basic). In one aspect, xylanase, a mannanase and/or aglucanase activity of polypeptides encoded by nucleic acids of theinvention retain activity at a temperature of at least about 80° C., 81°C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90°C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99°C., 100° C., 101° C., 102° C., 103° C., 103.5° C., 104° C., 105° C.,107° C., 108° C., 109° C. or 110° C., or more, and a basic pH of atleast about pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11,pH 11.5, pH 12, pH 12.5 or more (more basic).

The invention provides expression cassettes, cloning vehicles, or avector (e.g., expression vectors) comprising a nucleic acid comprising asequence of the invention. The cloning vehicle can comprise a viralvector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, abacteriophage or an artificial chromosome. The viral vector can comprisean adenovirus vector, a retroviral vector or an adeno-associated viralvector. The cloning vehicle can comprise an artificial chromosomecomprising a bacterial artificial chromosome (BAC), a bacteriophageP1-derived vector (PAC), a yeast artificial chromosome (YAC), or amammalian artificial chromosome (MAC).

The invention provides nucleic acid probes for identifying a nucleicacid encoding a polypeptide with a xylanase, a mannanase and/or aglucanase activity, wherein the probe comprises at least about 10, 15,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100, 125, 150, 175, 200,225, 250, 275, 300 or more consecutive bases of a nucleic acidcomprising an exemplary sequence of the invention, or, any sequence ofthe invention (as defined herein), wherein in one aspect (optionally)the probe comprises an oligonucleotide comprising between at least about10 to 300, about 25 to 250, about 10 to 50, about 20 to 60, about 30 to70, about 40 to 80, about 60 to 100, or about 50 to 150 or moreconsecutive bases.

The invention provides amplification primer pairs for amplifying anucleic acid encoding a polypeptide having a xylanase, a mannanaseand/or a glucanase activity, wherein the primer pair is capable ofamplifying a nucleic acid comprising an exemplary sequence of theinvention, or, any sequence of the invention (as defined herein), or asubsequence thereof, wherein optionally a member of the amplificationprimer sequence pair comprises an oligonucleotide comprising at leastabout 10 to 50 consecutive bases of the sequence, or, about 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35 or more consecutive bases of the sequence. Theinvention provides amplification primer pairs wherein the primer paircomprises a first member having a sequence as set forth by about thefirst (the 5′) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more residues of anexemplary sequence of the invention, or, any sequence of the invention(as defined herein), and a second member having a sequence as set forthby about the first (the 5′) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or moreresidues of the complementary strand of the first member.

The invention provides xylanase- and/or a glucanase-encoding nucleicacids generated by amplification of a polynucleotide using anamplification primer pair of the invention, wherein optionally theamplification is by polymerase chain reaction (PCR). In one aspect, thenucleic acid is generated by amplification of a gene library, wherein inone aspect (optionally) the gene library is an environmental library.The invention provides isolated, synthetic or recombinant xylanasesand/or a glucanases encoded by a xylanase- and/or a glucanase-encodingnucleic acid generated by amplification of a polynucleotide using anamplification primer pair of the invention. The invention providesmethods of amplifying a nucleic acid encoding a polypeptide having axylanase, a mannanase and/or a glucanase activity, the methodscomprising the step of amplification of a template nucleic acid with anamplification primer sequence pair capable of amplifying an exemplarysequence of the invention, or, any sequence of the invention (as definedherein), or a subsequence thereof.

The invention provides expression cassette, a vector or a cloningvehicle comprising a nucleic acid comprising a sequence of theinvention, wherein optionally the cloning vehicle comprises a viralvector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, abacteriophage or an artificial chromosome. The viral vector can comprisean adenovirus vector, a retroviral vector or an adeno-associated viralvector, or, the artificial chromosome comprises a bacterial artificialchromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeastartificial chromosome (YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cells comprising a nucleic acid orvector of the invention, or an expression cassette or cloning vehicle ofthe invention. The transformed cell can be a bacterial cell, a mammaliancell, a fungal cell, a yeast cell, an insect cell or a plant cell.

The invention provides transgenic non-human animals comprising asequence of the invention. The transgenic non-human animal can be amouse, a rat, a rabbit, a sheep, a pig, a chicken, a goat, a fish, adog, or a cow. The invention provides transgenic plants comprising asequence of the invention, e.g., wherein the plant is a corn plant, asorghum plant, a potato plant, a tomato plant, a wheat plant, an oilseedplant, a rapeseed plant, a soybean plant, a rice plant, a barley plant,a grass, or a tobacco plant. The invention provides transgenic seedscomprising a sequence of the invention, e.g., wherein the seed is a cornseed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palmkernel, a sunflower seed, a sesame seed, a rice, a barley, a peanut or atobacco plant seed.

The invention provides antisense oligonucleotides comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a sequence of the invention (including, e.g., exemplarysequences of the invention), or a subsequence thereof, whereinoptionally the antisense oligonucleotide is between about 10 to 50,about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 basesin length, and in one aspect (optionally) the stringent conditionscomprise a wash step comprising a wash in 0.2×SSC at a temperature ofabout 65° C. for about 15 minutes.

The invention provides methods of inhibiting the translation of axylanase, a mannanase and/or a glucanase message in a cell comprisingadministering to the cell or expressing in the cell an antisenseoligonucleotide comprising a nucleic acid sequence complementary to orcapable of hybridizing under stringent conditions to a sequence of theinvention (including, e.g., exemplary sequences of the invention).

The invention provides double-stranded inhibitory RNA (RNAi) moleculescomprising a subsequence of a sequence of the invention (including,e.g., exemplary sequences of the invention). The double-strandedinhibitory RNA (RNAi) molecule can be about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more duplexnucleotides in length. The invention provides methods of inhibiting theexpression of a xylanase, a mannanase and/or a glucanase in a cellcomprising administering to the cell or expressing in the cell adouble-stranded inhibitory RNA (iRNA), wherein the RNA comprises asubsequence of a sequence of the invention (including, e.g., exemplarysequences of the invention).

The invention provides isolated, synthetic or recombinant polypeptideshaving a xylanase, a mannanase and/or a glucanase activity, orpolypeptides capable of generating an immune response specific for axylanase (e.g., an endoxylanase), a mannanase and/or a glucanase (e.g.,an epitope); and in alternative aspects peptide and polypeptide of theinvention comprise a sequence:

(a) comprising an amino acid sequence having at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or has 100% (complete) sequenceidentity to:

(i) the amino acid sequence of SEQ ID NO:2, or enzymatically activefragments thereof, and having at least one amino acid residue change (orthe equivalent thereof) as set forth in Table 1, or having at least one,two, three, four, five, six, seven, eight, nine, ten, eleven, twelve,thirteen, fourteen, fifteen, sixteen, seventeen or eighteen or some orall of the following amino acid residue changes: amino acid residue 4 ischanged from a T (or thr, or threonine) to an N (or asn, or asparagine);amino acid residue 4 is changed from a T (or thr, or threonine) to an R(or arg, or arginine); amino acid residue 4 is changed from a T (or thr,or threonine) to an H (or his, or histidine); amino acid residue 9 ischanged from a P (or pro, or proline) to an D (or asp, or asparticacid); amino acid residue 17 is changed from a F (or phe, orphenylalanine) to an V (or val, or valine); amino acid residue 21 ischanged from a F (or phe, or phenylalanine) to an Y (or tyr, ortyrosine); amino acid residue 33 is changed from a L (or leu, orleucine) to an A (or ala, or alanine); amino acid residue 38 is changedfrom a R (or arg, or arginine) to an H (or his, or histidine); aminoacid residue 44 is changed from a S (or ser, or serine) to an T (or thr,or threonine); amino acid residue 63 is changed from a I (or ile, orisoleucine) to an V (or val, or valine); amino acid residue 73 is changefrom a G (or gly, or glycine) to an Y (or tyr, or tyrosine); amino acidresidue 73 is changed from a G (or gly, or glycine) to an V (or val, orvaline); amino acid residue 73 is changed from a G (or gly, or glycine)to an E (or glu, or glutamic acid); amino acid residue 108 is changedfrom a F (or phe, or phenylalaine) to an K (or lys, or lysine); aminoacid residue 125 is change from a Q (or gln, or glutamine) to an Y (ortyr, or tyrosine); amino acid residue 150 is change from a V (or val, orvaline) to an A (or ala, or alanine); amino acid residue 188 is changedfrom a S (or ser, or serine) to an E (or glu, or glutamic acid); and/oramino acid residue 189 is changed from a S (or ser, or serine) to an Q(or gln, or glutamine), or

(ii) the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24;

wherein the polypeptide or peptide of (i) or (ii) has a xylanase, amannanase and/or a glucanase activity, or the polypeptide or peptide iscapable of generating a xylanase, a mannanase and/or a glucanasespecific antibody (a polypeptide or peptide that acts as an epitope orimmunogen),

(b) the polypeptide or peptide of (a), wherein the sequence identitiesare determined: (A) by analysis with a sequence comparison algorithm orby a visual inspection, or (B) over a region of at least about 20, 25,30, 35, 40, 45, 50, 55, 60, 75, 100, 150, 200, 250, 300 or more aminoacid residues, or over the full length of the polypeptide or peptide orenzyme, and/or enzymatically active subsequences (fragments) thereof,

(c) the polypeptide or peptide of (a) of (b), wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection, and optionally the sequencecomparison algorithm is a BLAST version 2.2.2 algorithm where afiltering setting is set to blastall -p blastp -d “nr pataa”-F F, andall other options are set to default;

(d) an amino acid sequence encoded by the nucleic acid of claim 1,wherein the polypeptide has (i) a xylanase, a mannanase and/or aglucanase activity, or, (ii) has immunogenic activity in that it iscapable of generating an antibody that specifically binds to apolypeptide having a sequence of (a), and/or enzymatically activesubsequences (fragments) thereof;

(e) the amino acid sequence of any of (a) to (d), and comprising atleast one amino acid residue conservative substitution, and thepolypeptide or peptide retains xylanase, a mannanase and/or a glucanaseactivity;

(e) the amino acid sequence of (e), wherein the conservativesubstitution comprises replacement of an aliphatic amino acid withanother aliphatic amino acid; replacement of a serine with a threonineor vice versa; replacement of an acidic residue with another acidicresidue; replacement of a residue bearing an amide group with anotherresidue bearing an amide group; exchange of a basic residue with anotherbasic residue; or, replacement of an aromatic residue with anotheraromatic residue, or a combination thereof,

(f) the amino acid sequence of (e), wherein the aliphatic residuecomprises Alanine, Valine, Leucine, Isoleucine or a synthetic equivalentthereof; the acidic residue comprises Aspartic acid, Glutamic acid or asynthetic equivalent thereof; the residue comprising an amide groupcomprises Aspartic acid, Glutamic acid or a synthetic equivalentthereof; the basic residue comprises Lysine, Arginine or a syntheticequivalent thereof; or, the aromatic residue comprises Phenylalanine,Tyrosine or a synthetic equivalent thereof;

(g) the polypeptide of any of (a) to (f) having a xylanase, a mannanaseand/or a glucanase activity but lacking a signal sequence, a preprodomain, a dockerin domain, and/or a carbohydrate binding module (CBM),

(h) the polypeptide of (g) wherein the carbohydrate binding module (CBM)comprises, or consists of, a xylan binding module, a cellulose bindingmodule, a lignin binding module, a xylose binding module, a mannansebinding module, a xyloglucan-specific module and/or aarabinofuranosidase binding module;

(i) the polypeptide of any of (a) to (h) having a xylanase, a mannanaseand/or a glucanase activity further comprising a heterologous sequence;

(j) the polypeptide of (i), wherein the heterologous sequence comprises,or consists of: (A) a heterologous signal sequence, a heterologouscarbohydrate binding module, a heterologous dockerin domain, aheterologous catalytic domain (CD), or a combination thereof; (B) thesequence of (A), wherein the heterologous signal sequence, carbohydratebinding module or catalytic domain (CD) is derived from a heterologouslignocellulosic enzyme; and/or, (C) a tag, an epitope, a targetingpeptide, a cleavable sequence, a detectable moiety or an enzyme;

(k) the polypeptide of (i) or (j), wherein the heterologous sequence orthe heterologous carbohydrate binding module (CBM) comprises, orconsists of, a xylan binding module, a cellulose binding module, alignin binding module, a xylose binding module, a mannan binding module,a xyloglucan-specific module and/or a arabinofuranosidase bindingmodule;

(1) polypeptide of (i), wherein the heterologous signal sequence targetsthe encoded protein to a vacuole, the endoplasmic reticulum, achloroplast or a starch granule; or

(m) comprising an amino acid sequence encoded any nucleic acid sequenceof this invention.

In one aspect, the isolated, synthetic or recombinant peptides of theinvention have a xylanase activity, e.g., wherein the xylanase activitycomprises catalyzing hydrolysis of internal β-1,4-xylosidic linkages;comprises an endo-1,4-beta-xylanase activity; comprises hydrolyzing axylan or an arabinoxylan to produce a smaller molecular weight xyloseand xylo-oligomer; comprises hydrolyzing a polysaccharide comprising a1,4β-glycoside-linked D-xylopyranose; comprises hydrolyzing a celluloseor a hemicellulose; comprises hydrolyzing a cellulose or a hemicellulosein a wood, wood product, paper pulp, paper product or paper waste;comprises catalyzing hydrolysis of a xylan or an arabinoxylan in a feedor a food product; or, comprises catalyzing hydrolysis of a xylan or anarabinoxylan in a microbial cell or a plant cell. The xylan cancomprises an arabinoxylan, e.g., a water soluble arabinoxylan, e.g., awater soluble arabinoxylan in a dough or a bread product.

In one aspect, the xylanase, a mannanase and/or a glucanase activitycomprises hydrolyzing polysaccharides, for example, comprising1,4-β-glycoside-linked D-xylopyranoses, or hydrolyzing hemicelluloses,e.g., hydrolyzing hemicelluloses in a wood, wood product, paper pulp,paper product or paper waste.

In one aspect, the xylanase, a mannanase and/or a glucanase activitycomprises catalyzing hydrolysis of polysaccharides, e.g., xylans, in afeed or a food product, such as a cereal-based animal feed, a wort or abeer, a milk or a milk product, a fruit or a vegetable. In one aspect,the xylanase activity comprises catalyzing hydrolysis of xylans in amicrobial cell or a plant cell.

The invention provides isolated, synthetic or recombinant polypeptidescomprising a polypeptide of the invention and lacking a signal sequenceor a prepro sequence. The invention provides isolated, synthetic orrecombinant polypeptides comprising a polypeptide of the invention andhaving a heterologous signal sequence or a heterologous prepro sequence.

In one aspect, a polypeptide of the invention has xylanase, a mannanaseand/or a glucanase activity comprising a specific activity at about 37°C. in the range from about 100 to about 1000 units per milligram ofprotein, from about 500 to about 750 units per milligram of protein,from about 500 to about 1200 units per milligram of protein, or fromabout 750 to about 1000 units per milligram of protein. In one aspect,units are defined as 0.1 to 20 units/g of pulp, where a unit equals umolof xylose released per minute per mg of enzyme, using arabinoxylan as asubstrate as described in the Nelson Somogyi assay, described in detailbelow. In alternative aspects, polypeptides of the invention havexylanase, a mannanase and/or a glucanase activity in the range ofbetween about 0.05 to 20 units per gram of pulp, or 0.05, 0.10, 0.20,0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 or more units per gram of pulp (where a unit equals umol ofxylose released per minute per mg of enzyme, using arabinoxylan as asubstrate as described in the Nelson Somogyi assay).

In one aspect, the thermotolerance comprises retention of at least halfof the specific activity of the xylanase, a mannanase and/or a glucanaseat 37° C. after being heated to an elevated temperature, such as atemperature from about 0° C. to about 20° C., about 20° C. to about 37°C., about 37° C. to about 50° C., about 50° C. to about 70° C., about70° C. to about 75° C., about 75° C. to about 80° C., about 80° C. toabout 85° C., about 85° C. to about 90° C., about 90° C. to about 95°C., about 95° C. to about 100° C., about 100° C. to about 110° C., orhigher. The thermotolerance can comprise retention of specific activityat 37° C. in the range from about 500 to about 1200 units per milligramof protein after being heated to an elevated temperature, such as atemperature from about 0° C. to about 20° C., about 20° C. to about 37°C., about 37° C. to about 50° C., about 50° C. to about 70° C., about70° C. to about 75° C., about 75° C. to about 80° C., about 80° C. toabout 85° C., about 85° C. to about 90° C., about 90° C. to about 95°C., about 95° C. to about 100° C., about 100° C. to about 110° C., orhigher.

In one aspect, the polypeptides of the invention comprise at least oneglycosylation site or further comprises a polysaccharide. Theglycosylation can be an N-linked glycosylation, e.g., wherein thepolypeptide is glycosylated after being expressed in a P. pastoris or aS. pombe.

In one aspect, the xylanase, a mannanase and/or a glucanase activity ofpolypeptides of the invention retain activity under acidic conditionscomprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5 or pH 4 or less(more acidic), or, retain a xylanase, a mannanase and/or a glucanaseactivity after exposure to acidic conditions comprising about pH 6.5, pH6, pH 5.5, pH 5, pH 4.5 or pH 4 or less (more acidic); or, retainactivity under basic conditions comprising about pH 7, pH 7.5 pH 8.0, pH8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 ormore (more basic) or, retain a xylanase, a mannanase and/or a glucanaseactivity after exposure to basic conditions comprising about pH 7, pH7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12,pH 12.5 or more (more basic). In one aspect, xylanase, a mannanaseand/or a glucanase activity of polypeptides of the invention retainactivity at a temperature of at least about 80° C., 81° C., 82° C., 83°C., 84° C., 85° C., 86° C., 87° C., 88° C., 89° C. or 90° C., and abasic pH of at least about pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10,pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (more basic).

The invention provides protein preparation comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, aslurry, a solid or a gel. The invention provides heterodimers comprisinga polypeptide of the invention and a second domain. The second domaincan be a polypeptide and the heterodimer is a fusion protein. the seconddomain can be an epitope or a tag. The invention provides homodimers orheterodimers comprising a polypeptide of the invention. The inventionprovides immobilized polypeptides, wherein the polypeptide comprises asequence of the invention, or a subsequence thereof, or a polypeptideencoded by a nucleic acid of the invention, or a polypeptide comprisinga polypeptide of the invention and a second domain, e.g., wherein thepolypeptide is immobilized on or inside a cell, a vesicle, a liposome, afilm, a membrane, a metal, a resin, a polymer, a ceramic, a glass, amicroelectrode, a graphitic particle, a bead, a gel, a plate, an array,a capillary tube, a crystal, a tablet, a pill, a capsule, a powder, anagglomerate, a surface, a porous structure, or materials such as woodchips, brownstock, pulp, paper, and materials deriving therefrom.

The xylanases and/or a glucanases of the invention can be used orformulated alone or as mixture (a “cocktail”) of xylanases and/or aglucanases, and other hydrolytic enzymes such as cellulases, mannanases,proteases, lipases, amylases, or redox enzymes such as laccases,peroxidases, catalases, oxidases, or reductases. They can be usedformulated in a solid form such as a powder, a lyophilized preparation,a granule, a tablet, a bar, a crystal, a capsule, a pill, a pellet, orin a liquid form such as in an aqueous solution, an aerosol, a gel, apaste, a slurry, an aqueous/oil emulsion, a cream, a capsule, or in avesicular or micellar suspension. The formulations of the invention cancomprise any or a combination of the following ingredients: polyols suchas a polyethylene glycol, a polyvinylalcohol, a glycerol, a sugar suchas a sucrose, a sorbitol, a trehalose, a glucose, a fructose, a maltose,a mannose, a gelling agent such as a guar gum, a carageenan, analginate, a dextrans, a cellulosic derivative, a pectin, a salt such asa sodium chloride, a sodium sulfate, an ammonium sulfate, a calciumchloride, a magnesium chloride, a zinc chloride, a zinc sulfate, a saltof a fatty acid and a fatty acid derivative, a metal chelator such as anEDTA, an EGTA, a sodium citrate, an antimicrobial agent such as a fattyacid or a fatty acid derivative, a paraben, a sorbate, a benzoate, anadditional modulating compound to block the impact of an enzyme such asa protease, a bulk proteins such as a BSA, a wheat hydrolysate, a boratecompound, an amino acid or a peptide, an appropriate pH or temperaturemodulating compound, an emulsifier such as a non-ionic and/or an ionicdetergent, a redox agent such as a cystine/cysteine, a glutathione, anoxidized glutathione, a reduced or an antioxidant compound such as anascorbic acid, or a dispersant. Cross-linking and protein modificationsuch as pegylation, fatty acid modification, glycosylation can also beused to improve enzyme stability.

The invention provides arrays comprising immobilized polypeptide(s)and/or nucleic acids of the invention, and arrays comprising animmobilized oligonucleotide of the invention. The enzymes, fragmentsthereof and nucleic acids which encode the enzymes, or probes of theinvention, and fragments thereof, can be affixed to a solid support; andthese embodiments can be economical and efficient in the use of enzymesand nucleic acids of the invention in industrial, medical, research,pharmaceutical, food and feed and food and feed supplement processingand other applications and processes. For example, a consortium orcocktail of enzymes (or active fragments thereof), which are used in aspecific chemical reaction, can be attached to a solid support anddunked into a process vat. The enzymatic reaction can occur. Then, thesolid support can be taken out of the vat, along with the enzymesaffixed thereto, for repeated use. In one embodiment of the invention,the isolated nucleic acid is affixed to a solid support. In anotherembodiment of the invention, the solid support is selected from thegroup of a gel, a resin, a polymer, a ceramic, a glass, a microelectrodeand any combination thereof.

For example, solid supports useful in this invention include gels. Someexamples of gels include sepharose, gelatin, glutaraldehyde,chitosan-treated glutaraldehyde, albumin-glutaraldehyde,chitosan-Xanthan, toyopearl gel (polymer gel), alginate,alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magneticagarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEGhydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.Another solid support useful in the present invention are resins orpolymers. Some examples of resins or polymers include cellulose,acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50,polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.Another type of solid support useful in the present invention isceramic. Some examples include non-porous ceramic, porous ceramic, SiO2,Al2O3. Another type of solid support useful in the present invention isglass. Some examples include non-porous glass, porus glass, aminopropylglass or any combination thereof. Another type of solid support whichcan be used is a mcroelectrode. An example is a polyethyleneimine-coatedmagnetite. Graphitic particles can be used as a solid support. Anotherexample of a solid support is a cell, such as a red blood cell.

There are many methods which would be known to one of skill in the artfor immobilizing enzymes or fragments thereof, or nucleic acids, onto asolid support. Some examples of such methods include electrostaticdroplet generation, electrochemical means, via adsorption, via covalentbinding, via cross-linking, via a chemical reaction or process, viaencapsulation, via entrapment, via calcium alginate, or via poly(2-hydroxyethyl methacrylate). Like methods are described in Methods inEnzymology, Immobilized Enzymes and Cells, Part C. 1987. Academic Press.Edited by S. P. Colowick and N, O. Kaplan. Volume 136; andImmobilization of Enzymes and Cells. 1997. Humana Press. Edited by G. F.Bickerstaff. Series: Methods in Biotechnology, Edited by J. M. Walker.

The invention provides isolated, synthetic or recombinant antibodiesthat specifically binds to a polypeptide of the invention. The antibodycan be a monoclonal or a polyclonal antibody, or is a single chainedantibody. The invention provides hybridomas comprising an antibody thatspecifically binds to a polypeptide of the invention.

The invention provides methods of isolating or identifying a polypeptidewith a xylanase, a mannanase and/or a glucanase activity comprising thesteps of: (a) providing an antibody of the invention; (b) providing asample comprising polypeptides; and (c) contacting the sample of step(b) with the antibody of step (a) under conditions wherein the antibodycan specifically bind to the polypeptide, thereby isolating oridentifying a polypeptide having a xylanase, a mannanase and/or aglucanase activity. The invention provides methods of making ananti-xylanase and/or anti-glucanase antibody comprising administering toa non-human animal a nucleic acid of the invention or a subsequencethereof in an amount sufficient to generate a humoral immune response,thereby making an anti-xylanase and/or anti-glucanase antibody. Theinvention provides methods of making an anti-xylanase and/oranti-glucanase antibody comprising administering to a non-human animal apolypeptide of the invention or a subsequence thereof in an amountsufficient to generate a humoral immune response, thereby making ananti-xylanase and/or anti-glucanase antibody.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid operably linked toa promoter, wherein the nucleic acid comprises a sequence of theinvention; and (b) expressing the nucleic acid of step (a) underconditions that allow expression of the polypeptide, thereby producing arecombinant polypeptide. The method can further comprise transforming ahost cell with the nucleic acid of step (a) followed by expressing thenucleic acid of step (a), thereby producing a recombinant polypeptide ina transformed cell.

The invention provides methods for identifying a polypeptide having axylanase, a mannanase and/or a glucanase activity comprising: (a)providing a polypeptide of the invention; (b) providing a xylanase, amannanase and/or a glucanase substrate; and (c) contacting thepolypeptide with the substrate of step (b) and detecting a decrease inthe amount of substrate or an increase in the amount of a reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of the reaction product detects a polypeptidehaving a xylanase, a mannanase and/or a glucanase activity.

The invention provides methods for identifying a xylanase, a mannanaseand/or a glucanase substrate comprising: (a) providing a polypeptide ofthe invention; (b) providing a test substrate; and (c) contacting thepolypeptide of step (a) with the test substrate of step (b) anddetecting a decrease in the amount of substrate or an increase in theamount of reaction product, wherein a decrease in the amount of thesubstrate or an increase in the amount of a reaction product identifiesthe test substrate as a xylanase, a mannanase and/or a glucanasesubstrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising: (a) expressing a nucleicacid or a vector comprising the nucleic acid under conditions permissivefor translation of the nucleic acid to a polypeptide, wherein thenucleic acid has a sequence of the invention; (b) providing a testcompound; (c) contacting the polypeptide with the test compound; and (d)determining whether the test compound of step (b) specifically binds tothe polypeptide.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising: (a) providing apolypeptide of the invention; (b) providing a test compound; (c)contacting the polypeptide with the test compound; and (d) determiningwhether the test compound of step (b) specifically binds to thepolypeptide.

The invention provides methods for identifying a modulator of axylanase, a mannanase and/or a glucanase activity comprising: (a)providing a polypeptide of the invention; (b) providing a test compound;(c) contacting the polypeptide of step (a) with the test compound ofstep (b) and measuring an activity of the xylanase, a mannanase and/or aglucanase, wherein a change in the xylanase, a mannanase and/or aglucanase activity measured in the presence of the test compoundcompared to the activity in the absence of the test compound provides adetermination that the test compound modulates the xylanase, a mannanaseand/or a glucanase activity. The xylanase, a mannanase and/or aglucanase activity can be measured by providing a xylanase, a mannanaseand/or a glucanase substrate and detecting a decrease in the amount ofthe substrate or an increase in the amount of a reaction product, or, anincrease in the amount of the substrate or a decrease in the amount of areaction product. In one aspect, a decrease in the amount of thesubstrate or an increase in the amount of the reaction product with thetest compound as compared to the amount of substrate or reaction productwithout the test compound identifies the test compound as an activatorof a xylanase, a mannanase and/or a glucanase activity. In one aspect,an increase in the amount of the substrate or a decrease in the amountof the reaction product with the test compound as compared to the amountof substrate or reaction product without the test compound identifiesthe test compound as an inhibitor of a xylanase, a mannanase and/or aglucanase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence, wherein thepolypeptide sequence comprises sequence of the invention, a polypeptideencoded by a nucleic acid of the invention. The computer systems canfurther comprise a sequence comparison algorithm and a data storagedevice having at least one reference sequence stored thereon. In anotheraspect, the sequence comparison algorithm comprises a computer programthat indicates polymorphisms. In one aspect, the computer system canfurther comprise an identifier that identifies one or more features insaid sequence. The invention provides computer readable media havingstored thereon a polypeptide sequence or a nucleic acid sequence of theinvention. The invention provides methods for identifying a feature in asequence comprising the steps of: (a) reading the sequence using acomputer program which identifies one or more features in a sequence,wherein the sequence comprises a polypeptide sequence or a nucleic acidsequence of the invention; and (b) identifying one or more features inthe sequence with the computer program. The invention provides methodsfor comparing a first sequence to a second sequence comprising the stepsof: (a) reading the first sequence and the second sequence through useof a computer program which compares sequences, wherein the firstsequence comprises a polypeptide sequence or a nucleic acid sequence ofthe invention; and (b) determining differences between the firstsequence and the second sequence with the computer program. The step ofdetermining differences between the first sequence and the secondsequence can further comprise the step of identifying polymorphisms. Inone aspect, the method can further comprise an identifier thatidentifies one or more features in a sequence. In another aspect, themethod can comprise reading the first sequence using a computer programand identifying one or more features in the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a xylanase, a mannanase and/or aglucanase activity from an environmental sample comprising the steps of:(a) providing an amplification primer sequence pair for amplifying anucleic acid encoding a polypeptide having a xylanase, a mannanaseand/or a glucanase activity, wherein the primer pair is capable ofamplifying a nucleic acid of the invention; (b) isolating a nucleic acidfrom the environmental sample or treating the environmental sample suchthat nucleic acid in the sample is accessible for hybridization to theamplification primer pair; and, (c) combining the nucleic acid of step(b) with the amplification primer pair of step (a) and amplifyingnucleic acid from the environmental sample, thereby isolating orrecovering a nucleic acid encoding a polypeptide having a xylanase, amannanase and/or a glucanase activity from an environmental sample. Oneor each member of the amplification primer sequence pair can comprise anoligonucleotide comprising at least about 10 to 50 consecutive bases ofa sequence of the invention. In one aspect, the amplification primersequence pair is an amplification pair of the invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having a xylanase, a mannanase and/or aglucanase activity from an environmental sample comprising the steps of:(a) providing a polynucleotide probe comprising a nucleic acid of theinvention or a subsequence thereof; (b) isolating a nucleic acid fromthe environmental sample or treating the environmental sample such thatnucleic acid in the sample is accessible for hybridization to apolynucleotide probe of step (a); (c) combining the isolated nucleicacid or the treated environmental sample of step (b) with thepolynucleotide probe of step (a); and (d) isolating a nucleic acid thatspecifically hybridizes with the polynucleotide probe of step (a),thereby isolating or recovering a nucleic acid encoding a polypeptidehaving a xylanase, a mannanase and/or a glucanase activity from anenvironmental sample. The environmental sample can comprise a watersample, a liquid sample, a soil sample, an air sample or a biologicalsample. In one aspect, the biological sample can be derived from abacterial cell, a protozoan cell, an insect cell, a yeast cell, a plantcell, a fungal cell or a mammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having a xylanase, a mannanase and/or a glucanaseactivity comprising the steps of: (a) providing a template nucleic acidcomprising a nucleic acid of the invention; and (b) modifying, deletingor adding one or more nucleotides in the template sequence, or acombination thereof, to generate a variant of the template nucleic acid.In one aspect, the method can further comprise expressing the variantnucleic acid to generate a variant xylanase, a mannanase and/or aglucanase polypeptide. The modifications, additions or deletions can beintroduced by a method comprising error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (e.g., GeneReassembly, see, e.g., U.S. Pat.No. 6,537,776), Gene Site Saturation Mutagenesis (GS SM), syntheticligation reassembly (SLR) or a combination thereof. In another aspect,the modifications, additions or deletions are introduced by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until a xylanase,a mannanase and/or a glucanase having an altered or different activityor an altered or different stability from that of a polypeptide encodedby the template nucleic acid is produced. In one aspect, the variantxylanase, a mannanase and/or a glucanase polypeptide is thermotolerant,and retains some activity after being exposed to an elevatedtemperature. In another aspect, the variant xylanase, a mannanase and/ora glucanase polypeptide has increased glycosylation as compared to thexylanase, a mannanase and/or a glucanase encoded by a template nucleicacid. Alternatively, the variant xylanase, a mannanase and/or aglucanase polypeptide has a xylanase, a mannanase and/or a glucanaseactivity under a high temperature, wherein the xylanase, a mannanaseand/or a glucanase encoded by the template nucleic acid is not activeunder the high temperature. In one aspect, the method can be iterativelyrepeated until a xylanase, a mannanase and/or a glucanase codingsequence having an altered codon usage from that of the template nucleicacid is produced. In another aspect, the method can be iterativelyrepeated until a xylanase, a mannanase and/or a glucanase gene havinghigher or lower level of message expression or stability from that ofthe template nucleic acid is produced. In another aspect, formulation ofthe final xylanase, a mannanase and/or a glucanase product enables anincrease or modulation of the performance of the xylanase, a mannanaseand/or a glucanase in the product.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a xylanase, a mannanase and/or a glucanaseactivity to increase its expression in a host cell, the methodcomprising: (a) providing a nucleic acid of the invention encoding apolypeptide having a xylanase, a mannanase and/or a glucanase activity;and, (b) identifying a non-preferred or a less preferred codon in thenucleic acid of step (a) and replacing it with a preferred or neutrallyused codon encoding the same amino acid as the replaced codon, wherein apreferred codon is a codon over-represented in coding sequences in genesin the host cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell, therebymodifying the nucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a xylanase, a mannanase and/or a glucanaseactivity; the method comprising: (a) providing a nucleic acid of theinvention; and, (b) identifying a codon in the nucleic acid of step (a)and replacing it with a different codon encoding the same amino acid asthe replaced codon, thereby modifying codons in a nucleic acid encodinga xylanase, a mannanase and/or a glucanase.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having a xylanase, a mannanase and/or a glucanaseactivity to increase its expression in a host cell, the methodcomprising: (a) providing a nucleic acid of the invention encoding axylanase, a mannanase and/or a glucanase polypeptide; and, (b)identifying a non-preferred or a less preferred codon in the nucleicacid of step (a) and replacing it with a preferred or neutrally usedcodon encoding the same amino acid as the replaced codon, wherein apreferred codon is a codon over-represented in coding sequences in genesin the host cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell, therebymodifying the nucleic acid to increase its expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having a xylanase, a mannanase and/or a glucanaseactivity to decrease its expression in a host cell, the methodcomprising: (a) providing a nucleic acid of the invention; and (b)identifying at least one preferred codon in the nucleic acid of step (a)and replacing it with a non-preferred or less preferred codon encodingthe same amino acid as the replaced codon, wherein a preferred codon isa codon over-represented in coding sequences in genes in a host cell anda non-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to decrease its expression in a host cell. In one aspect,the host cell can be a bacterial cell, a fungal cell, an insect cell, ayeast cell, a plant cell or a mammalian cell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified xylanase, a mannanase and/or aglucanase active sites or substrate binding sites, wherein the modifiedactive sites or substrate binding sites are derived from a first nucleicacid comprising a sequence encoding a first active site or a firstsubstrate binding site the method comprising: (a) providing a firstnucleic acid encoding a first active site or first substrate bindingsite, wherein the first nucleic acid sequence comprises a sequence thathybridizes under stringent conditions to a sequence of the invention, ora subsequence thereof, and the nucleic acid encodes a xylanase, amannanase and/or a glucanase active site or a xylanase, a mannanaseand/or a glucanase substrate binding site; (b) providing a set ofmutagenic oligonucleotides that encode naturally-occurring amino acidvariants at a plurality of targeted codons in the first nucleic acid;and, (c) using the set of mutagenic oligonucleotides to generate a setof active site-encoding or substrate binding site-encoding variantnucleic acids encoding a range of amino acid variations at each aminoacid codon that was mutagenized, thereby producing a library of nucleicacids encoding a plurality of modified xylanase, a mannanase and/or aglucanase active sites or substrate binding sites. In one aspect, themethod comprises mutagenizing the first nucleic acid of step (a) by amethod comprising an optimized directed evolution system, Gene SiteSaturation Mutagenesis (GSSM), or a synthetic ligation reassembly (SLR).In one aspect, the method comprises mutagenizing the first nucleic acidof step (a) or variants by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (GeneReassembly, U.S. Pat. No. 6,537,776),Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly(SLR) and a combination thereof. In one aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprising:(a) providing a plurality of biosynthetic enzymes capable ofsynthesizing or modifying a small molecule, wherein one of the enzymescomprises a xylanase, a mannanase and/or a glucanase enzyme encoded by anucleic acid of the invention; (b) providing a substrate for at leastone of the enzymes of step (a); and (c) reacting the substrate of step(b) with the enzymes under conditions that facilitate a plurality ofbiocatalytic reactions to generate a small molecule by a series ofbiocatalytic reactions. The invention provides methods for modifying asmall molecule comprising: (a) providing a xylanase, a mannanase and/ora glucanase enzyme, wherein the enzyme comprises a polypeptide of theinvention, or, a polypeptide encoded by a nucleic acid of the invention,or a subsequence thereof; (b) providing a small molecule; and (c)reacting the enzyme of step (a) with the small molecule of step (b)under conditions that facilitate an enzymatic reaction catalyzed by thexylanase, a mannanase and/or a glucanase enzyme, thereby modifying asmall molecule by a xylanase, a mannanase and/or a glucanase enzymaticreaction. In one aspect, the method can comprise a plurality of smallmolecule substrates for the enzyme of step (a), thereby generating alibrary of modified small molecules produced by at least one enzymaticreaction catalyzed by the xylanase, a mannanase and/or a glucanaseenzyme. In one aspect, the method can comprise a plurality of additionalenzymes under conditions that facilitate a plurality of biocatalyticreactions by the enzymes to form a library of modified small moleculesproduced by the plurality of enzymatic reactions. In another aspect, themethod can further comprise the step of testing the library to determineif a particular modified small molecule that exhibits a desired activityis present within the library. The step of testing the library canfurther comprise the steps of systematically eliminating all but one ofthe biocatalytic reactions used to produce a portion of the plurality ofthe modified small molecules within the library by testing the portionof the modified small molecule for the presence or absence of theparticular modified small molecule with a desired activity, andidentifying at least one specific biocatalytic reaction that producesthe particular modified small molecule of desired activity.

The invention provides methods for determining a functional fragment ofa xylanase, a mannanase and/or a glucanase enzyme comprising the stepsof: (a) providing a xylanase, a mannanase and/or a glucanase enzyme,wherein the enzyme comprises a polypeptide of the invention, or apolypeptide encoded by a nucleic acid of the invention, or a subsequencethereof; and (b) deleting a plurality of amino acid residues from thesequence of step (a) and testing the remaining subsequence for axylanase, a mannanase and/or a glucanase activity, thereby determining afunctional fragment of a xylanase, a mannanase and/or a glucanaseenzyme. In one aspect, the xylanase, a mannanase and/or a glucanaseactivity is measured by providing a xylanase, a mannanase and/or aglucanase substrate and detecting a decrease in the amount of thesubstrate or an increase in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising: (a) making a modified cell by modifying the geneticcomposition of a cell, wherein the genetic composition is modified byaddition to the cell of a nucleic acid of the invention; (b) culturingthe modified cell to generate a plurality of modified cells; (c)measuring at least one metabolic parameter of the cell by monitoring thecell culture of step (b) in real time; and, (d) analyzing the data ofstep (c) to determine if the measured parameter differs from acomparable measurement in an unmodified cell under similar conditions,thereby identifying an engineered phenotype in the cell using real-timemetabolic flux analysis. In one aspect, the genetic composition of thecell can be modified by a method comprising deletion of a sequence ormodification of a sequence in the cell, or, knocking out the expressionof a gene. In one aspect, the method can further comprise selecting acell comprising a newly engineered phenotype. In another aspect, themethod can comprise culturing the selected cell, thereby generating anew cell strain comprising a newly engineered phenotype.

The invention provides isolated, synthetic or recombinant signalsequences consisting of, or comprising, a sequence as set forth inresidues 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18,1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26,1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34,1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43or 1 to 44, of a polypeptide of the invention, including exemplarypolypeptide sequences of the invention.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising a signal peptide (SP) and at least a second domaincomprising a heterologous polypeptide or peptide comprising a sequenceof the invention, or a subsequence thereof, wherein the heterologouspolypeptide or peptide is not naturally associated with the signalpeptide (SP). In one aspect, the signal peptide (SP) is not derived froma xylanase, a mannanase and/or a glucanase. The heterologous polypeptideor peptide can be amino terminal to, carboxy terminal to or on both endsof the signal peptide (SP) or a xylanase, a mannanase and/or a glucanasecatalytic domain (CD). The invention provides isolated, synthetic orrecombinant nucleic acids encoding a chimeric polypeptide, wherein thechimeric polypeptide comprises at least a first domain comprising signalpeptide (SP) and at least a second domain comprising a heterologouspolypeptide or peptide comprising a sequence of the invention, or asubsequence thereof, wherein the heterologous polypeptide or peptide isnot naturally associated with the signal peptide (SP).

The invention provides methods of increasing thermotolerance orthermostability of a xylanase, a mannanase and/or a glucanasepolypeptide, the method comprising glycosylating a xylanase, a mannanaseand/or a glucanase polypeptide, wherein the polypeptide comprises atleast thirty contiguous amino acids of a polypeptide of the invention;or a polypeptide encoded by a nucleic acid sequence of the invention,thereby increasing the thermotolerance or thermostability of thexylanase, a mannanase and/or a glucanase polypeptide. In one aspect, thexylanase, a mannanase and/or a glucanase specific activity can bethermostable or thermotolerant at a temperature in the range fromgreater than about 0° C. to about 20° C., about 20° C. to about 37° C.,about 37° C. to about 50° C., about 50° C. to about 70° C., about 70° C.to about 75° C., about 75° C. to about 80° C., about 80° C. to about 85°C., about 85° C. to about 90° C., about 90° C. to about 95° C., about95° C. to about 100° C., about 100° C. to about 110° C., or higher.

The invention provides methods for overexpressing a recombinantxylanase, a mannanase and/or a glucanase polypeptide in a cellcomprising expressing a vector comprising a nucleic acid comprising anucleic acid of the invention or a nucleic acid sequence of theinvention, wherein the sequence identities are determined by analysiswith a sequence comparison algorithm or by visual inspection, whereinoverexpression is effected by use of a high activity promoter, adicistronic vector or by gene amplification of the vector.

The invention provides methods of making a transgenic plant and seedscomprising: (a) introducing a heterologous nucleic acid sequence intothe cell, wherein the heterologous nucleic sequence comprises a nucleicacid sequence of the invention, thereby producing a transformed plant orseed cell; and (b) producing a transgenic plant from the transformedcell or seed. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising: (a) transforming the plant cellwith a heterologous nucleic acid sequence operably linked to a promoter,wherein the heterologous nucleic sequence comprises a nucleic acid ofthe invention; (b) growing the plant under conditions wherein theheterologous nucleic acids sequence is expressed in the plant cell. Theinvention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising: (a) transforming the plant cellwith a heterologous nucleic acid sequence operably linked to a promoter,wherein the heterologous nucleic sequence comprises a sequence of theinvention; (b) growing the plant under conditions wherein theheterologous nucleic acids sequence is expressed in the plant cell.

The invention provides methods for hydrolyzing, breaking up ordisrupting a xylan-comprising composition comprising: (a) providing apolypeptide of the invention having a xylanase, a mannanase and/or aglucanase activity, or a polypeptide encoded by a nucleic acid of theinvention; (b) providing a composition comprising a xylan; and (c)contacting the polypeptide of step (a) with the composition of step (b)under conditions wherein the xylanase, a mannanase and/or a glucanasehydrolyzes, breaks up or disrupts the xylan-comprising composition. Inone aspect, the composition comprises a plant cell, a bacterial cell, ayeast cell, an insect cell, or an animal cell. Thus, the composition cancomprise any plant or plant part, any xylan-containing food or feed, awaste product and the like.

The invention provides methods for liquefying or removing axylan-comprising composition comprising: (a) providing a polypeptide ofthe invention having a xylanase activity, or a polypeptide encoded by anucleic acid of the invention; (b) providing a composition comprising axylan; and (c) contacting the polypeptide of step (a) with thecomposition of step (b) under conditions wherein the xylanase removes,softens or liquefies the xylan-comprising composition.

The invention provides detergent compositions comprising a polypeptideof the invention, or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide has a xylanase, a mannanase and/or aglucanase activity. The xylanase can be a nonsurface-active xylanase, amannanase and/or a glucanase or a surface-active xylanase, a mannanaseand/or a glucanase. The xylanase, a mannanase and/or a glucanase can beformulated in a non-aqueous liquid composition, a cast solid, a granularform, a particulate form, a compressed tablet, a gel form, a paste or aslurry form. The invention provides methods for washing an objectcomprising: (a) providing a composition comprising a polypeptide of theinvention having a xylanase, a mannanase and/or a glucanase activity, ora polypeptide encoded by a nucleic acid of the invention; (b) providingan object; and (c) contacting the polypeptide of step (a) and the objectof step (b) under conditions wherein the composition can wash theobject.

The invention provides textiles or fabrics, including, e.g., threads,comprising a polypeptide of the invention, or a polypeptide encoded by anucleic acid of the invention. In one aspect, the textiles or fabricscomprise xylan-containing fibers. The invention provides methods fortreating a textile or fabric (e.g., removing a stain from a composition)comprising: (a) providing a composition comprising a polypeptide of theinvention having a xylanase, a mannanase and/or a glucanase activity, ora polypeptide encoded by a nucleic acid of the invention; (b) providinga textile or fabric comprising a xylan; and (c) contacting thepolypeptide of step (a) and the composition of step (b) under conditionswherein the xylanase, a mannanase and/or a glucanase can treat thetextile or fabric (e.g., remove the stain). The invention providesmethods for improving the finish of a fabric comprising: (a) providing acomposition comprising a polypeptide of the invention having a xylanase,a mannanase and/or a glucanase activity, or a polypeptide encoded by anucleic acid of the invention; (b) providing a fabric; and (c)contacting the polypeptide of step (a) and the fabric of step (b) underconditions wherein the polypeptide can treat the fabric therebyimproving the finish of the fabric. In one aspect, the fabric is a woolor a silk. In another aspect, the fabric is a cellulosic fiber or ablend of a natural fiber and a synthetic fiber.

The invention provides feeds or foods comprising a polypeptide of theinvention, or a polypeptide encoded by a nucleic acid of the invention.The invention provides methods for hydrolyzing xylans in a feed or afood prior to consumption by an animal comprising: (a) obtaining a feedmaterial comprising a xylanase, a mannanase and/or a glucanase of theinvention, or a xylanase, a mannanase and/or a glucanase encoded by anucleic acid of the invention; and (b) adding the polypeptide of step(a) to the feed or food material in an amount sufficient for asufficient time period to cause hydrolysis of the xylan and formation ofa treated food or feed, thereby hydrolyzing the xylans in the food orthe feed prior to consumption by the animal. In one aspect, theinvention provides methods for hydrolyzing xylans in a feed or a foodafter consumption by an animal comprising: (a) obtaining a feed materialcomprising a xylanase, a mannanase and/or a glucanase of the invention,or a xylanase, a mannanase and/or a glucanase encoded by a nucleic acidof the invention; (b) adding the polypeptide of step (a) to the feed orfood material; and (c) administering the feed or food material to theanimal, wherein after consumption, the xylanase, a mannanase and/or aglucanase causes hydrolysis of xylans in the feed or food in thedigestive tract of the animal. The food or the feed can be, e.g., acereal, a grain, a corn and the like.

The invention provides dough or bread products comprising a polypeptidehaving a xylanase, a mannanase and/or a glucanase activity, wherein thepolypeptide comprises a sequence of the invention, or the polypeptide isencoded by a nucleic acid comprising a sequence of the invention, or anenzymatically active fragment thereof. The invention provides methods ofdough conditioning comprising contacting a dough or a bread product withat least one polypeptide having a xylanase, a mannanase and/or aglucanase activity, wherein the polypeptide comprises a sequence of theinvention, or the polypeptide is encoded by a nucleic acid comprising asequence of the invention, or an enzymatically active fragment thereof,under conditions sufficient for conditioning the dough.

The invention provides beverages comprising a polypeptide having axylanase, a mannanase and/or a glucanase activity, wherein thepolypeptide comprises a sequence of the invention, or the polypeptide isencoded by a nucleic acid comprising a sequence of the invention. Theinvention provides methods of beverage production comprisingadministration of at least one polypeptide having a xylanase, amannanase and/or a glucanase activity, wherein the polypeptide comprisesa sequence of the invention, or the polypeptide is encoded by a nucleicacid comprising a sequence of the invention, or an enzymatically activefragment thereof, to a beverage or a beverage precursor under conditionssufficient for decreasing the viscosity of the beverage, wherein in oneaspect (optionally) the beverage or beverage precursor is a wort or abeer.

The invention provides food or nutritional supplements for an animalcomprising a polypeptide of the invention, e.g., a polypeptide encodedby the nucleic acid of the invention. In one aspect, the polypeptide inthe food or nutritional supplement can be glycosylated. The inventionprovides edible enzyme delivery matrices comprising a polypeptide of theinvention, e.g., a polypeptide encoded by the nucleic acid of theinvention. In one aspect, the delivery matrix comprises a pellet. In oneaspect, the polypeptide can be glycosylated. In one aspect, thexylanase, a mannanase and/or a glucanase activity is thermotolerant. Inanother aspect, the xylanase, a mannanase and/or a glucanase activity isthermostable.

The invention provides a food, a feed or a nutritional supplementcomprising a polypeptide of the invention. The invention providesmethods for utilizing a xylanase, a mannanase and/or a glucanase as anutritional supplement in an animal diet, the method comprising:preparing a nutritional supplement containing a xylanase, a mannanaseand/or a glucanase enzyme comprising at least thirty contiguous aminoacids of a polypeptide of the invention; and administering thenutritional supplement to an animal to increase utilization of a xylancontained in a feed or a food ingested by the animal. The animal can bea human, a ruminant or a monogastric animal. The xylanase, a mannanaseand/or a glucanase enzyme can be prepared by expression of apolynucleotide encoding the xylanase, a mannanase and/or a glucanase inan organism selected from the group consisting of a bacterium, a yeast,a plant, an insect, a fungus and an animal. The organism can be selectedfrom the group consisting of an S. pombe, S. cerevisiae, Pichiapastoris, Pseudomonas sp., E. coli, Streptomyces sp., Bacillus sp. andLactobacillus sp.

The invention provides edible enzyme delivery matrix comprising athermostable recombinant xylanase, a mannanase and/or a glucanaseenzyme, e.g., a polypeptide of the invention. The invention providesmethods for delivering a xylanase, a mannanase and/or a glucanasesupplement to an animal, the method comprising: preparing an edibleenzyme delivery matrix in the form of pellets comprising a granulateedible carrier and a thermostable recombinant xylanase, a mannanaseand/or a glucanase enzyme, wherein the pellets readily disperse thexylanase, a mannanase and/or a glucanase enzyme contained therein intoaqueous media, and administering the edible enzyme delivery matrix tothe animal. The recombinant xylanase, a mannanase and/or a glucanaseenzyme can comprise a polypeptide of the invention. The granulate ediblecarrier can comprise a carrier selected from the group consisting of agrain germ, a grain germ that is spent of oil, a hay, an alfalfa, atimothy, a soy hull, a sunflower seed meal and a wheat midd. The ediblecarrier can comprise grain germ that is spent of oil. The xylanase, amannanase and/or a glucanase enzyme can be glycosylated to providethermostability at pelletizing conditions. The delivery matrix can beformed by pelletizing a mixture comprising a grain germ and a xylanase,a mannanase and/or a glucanase. The pelletizing conditions can includeapplication of steam. The pelletizing conditions can compriseapplication of a temperature in excess of about 80° C. for about 5minutes and the enzyme retains a specific activity of at least 350 toabout 900 units per milligram of enzyme.

The invention provides methods for improving texture and flavor of adairy product comprising: (a) providing a polypeptide of the inventionhaving a xylanase, a mannanase and/or a glucanase activity, or axylanase, a mannanase and/or a glucanase encoded by a nucleic acid ofthe invention; (b) providing a dairy product; and (c) contacting thepolypeptide of step (a) and the dairy product of step (b) underconditions wherein the xylanase, a mannanase and/or a glucanase canimprove the texture or flavor of the dairy product. In one aspect, thedairy product comprises a cheese or a yogurt. The invention providesdairy products comprising a xylanase, a mannanase and/or a glucanase ofthe invention, or is encoded by a nucleic acid of the invention.

The invention provides methods for improving the extraction of oil froman oil-rich plant material comprising: (a) providing a polypeptide ofthe invention having a xylanase, a mannanase and/or a glucanaseactivity, or a xylanase, a mannanase and/or a glucanase encoded by anucleic acid of the invention; (b) providing an oil-rich plant material;and (c) contacting the polypeptide of step (a) and the oil-rich plantmaterial. In one aspect, the oil-rich plant material comprises anoil-rich seed. The oil can be a soybean oil, an olive oil, a rapeseed(canola) oil or a sunflower oil.

The invention provides methods for preparing a fruit or vegetable juice,syrup, puree or extract comprising: (a) providing a polypeptide of theinvention having a xylanase, a mannanase and/or a glucanase activity, ora xylanase, a mannanase and/or a glucanase encoded by a nucleic acid ofthe invention; (b) providing a composition or a liquid comprising afruit or vegetable material; and (c) contacting the polypeptide of step(a) and the composition, thereby preparing the fruit or vegetable juice,syrup, puree or extract.

The invention provides papers or paper products or paper pulp comprisinga xylanase, a mannanase and/or a glucanase of the invention, or apolypeptide encoded by a nucleic acid of the invention. The inventionprovides methods for treating a biomass, e.g., any paper or a paper orwood pulp comprising: (a) providing a polypeptide of the inventionhaving a xylanase, a mannanase and/or a glucanase activity, or axylanase, a mannanase and/or a glucanase encoded by a nucleic acid ofthe invention; (b) providing a composition, e.g, a biomass, comprising apaper or a paper or wood pulp; and (c) contacting the polypeptide ofstep (a) and the composition of step (b) under conditions wherein thexylanase, a mannanase and/or a glucanase can treat the paper or paper orwood pulp.

The invention provides methods for reducing the amount of lignin(delignification), or solubilizing a lignin, in a paper or paperproduct, a paper waste, a wood, wood pulp or wood product, or a wood orpaper recycling composition, comprising contacting the paper or paperproduct, wood, wood pulp or wood product, or wood or paper recyclingcomposition with a polypeptide of the invention, or an enzymaticallyactive fragment thereof.

The invention provides methods for hydrolyzing hemicelluloses in a wood,wood product, paper pulp, paper product or paper waste comprisingcontacting the wood, wood product, paper pulp, paper product or paperwaste with a polypeptide of the invention, or an enzymatically activefragment thereof.

The invention provides methods for enzymatic decoloring (e.g.,bleaching) of paper, hemp or flax pulp comprising contacting the paper,hemp or flax pulp with a xylanase, a mannanase and/or a glucanase and adecoloring (e.g., bleaching) agent, wherein the xylanase, a mannanaseand/or a glucanase comprises a polypeptide of the invention, or anenzymatically active fragment thereof. The decoloring (e.g., bleaching)agent can comprise oxygen or hydrogen peroxide.

The invention provides methods for of decoloring (e.g., bleaching) alignocellulose pulp comprising contacting the lignocellulose pulp with axylanase, a mannanase and/or a glucanase, wherein the xylanase, amannanase and/or a glucanase comprises a polypeptide of the invention,or an enzymatically active fragment thereof.

The invention provides methods for enzymatic deinking of paper, paperwaste, paper recycled product, deinking toner from non-contact printedwastepaper or mixtures of non-contact and contact printed wastepaper,comprising contacting the paper, paper waste, paper recycled product,non-contact printed wastepaper or contact printed wastepaper with axylanase, a mannanase and/or a glucanase, wherein the xylanase, amannanase and/or a glucanase comprises a polypeptide of the invention,or an enzymatically active fragment thereof.

The invention provides methods for decoloring (e.g., bleaching) athread, fabric, yarn, cloth or textile comprising contacting the fabric,yarn, cloth or textile with a xylanase, a mannanase and/or a glucanaseunder conditions suitable to produce a whitening of the textile, whereinthe xylanase, a mannanase and/or a glucanase comprises a polypeptide ofthe invention, or an enzymatically active fragment thereof. The thread,fabric, yarn, cloth or textile can comprise a non-cotton cellulosicthread, fabric, yarn, cloth or textile. The invention provides fabrics,yarns, cloths or textiles comprising a polypeptide having a sequence ofthe invention, or a polypeptide encoded by a nucleic acid comprising asequence of the invention, or an enzymatically active fragment thereof,wherein in one aspect (optionally) the fabric, yarn, cloth or textilecomprises a non-cotton cellulosic fabric, yarn, cloth or textile.

The invention provides methods for decoloring (e.g., bleaching) ordeinking newspaper comprising contacting the newspaper, wherein thexylanase, a mannanase and/or a glucanase comprises a polypeptide of theinvention, or an enzymatically active fragment thereof.

The invention provides wood, wood chips, wood pulp, wood products, paperpulps, paper products, newspapers or paper waste comprising apolypeptide of the invention, or an enzymatically active fragmentthereof. The invention provides thread, fabric, yarn, cloth or textilecomprising a polypeptide of the invention, or an enzymatically activefragment thereof.

The invention provides methods for reducing lignin in a wood or woodproduct comprising contacting the wood or wood product with apolypeptide having a xylanase, a mannanase and/or a glucanase activity,wherein the polypeptide has a sequence of the invention, or thepolypeptide is encoded by a nucleic acid comprising a sequence of theinvention, or an enzymatically active fragment thereof.

The invention provides methods for reducing a lignin in a biomass, e.g.,in a wood, a wood pulp, a Kraft pulp, a paper, a paper product or apaper pulp under high temperature and basic pH conditions, the methodcomprising: (a) providing at least one polypeptide having a xylanase, amannanase and/or a glucanase activity, wherein the polypeptide retainsxylanase, a mannanase and/or a glucanase activity under conditionscomprising a temperature of at least about 80° C., 85° C., 90° C. ormore, and a basic pH of at least about pH 10.5, pH 11, pH 12, pH 12.5 ormore (basic) wherein the polypeptide comprises a xylanase, a mannanaseand/or a glucanase having a sequence of the invention, or the xylanase,a mannanase and/or a glucanase is encoded by a nucleic acid comprising asequence of the invention, or an enzymatically active fragment thereof;(b) providing a lignin-comprising biomass, e.g., a lignin-comprisingwood, wood pulp, Kraft pulp, paper, paper product or paper pulp; and (c)contacting the wood, wood pulp, Kraft pulp, paper, paper product orpaper pulp with the polypeptide of step (a) under conditions comprisinga temperature of at least about 80° C., 85° C., 90° C. or more, and abasic pH of at least about pH 10.5, pH 11, pH 12, pH 12.5 or more(basic), wherein the polypeptide reduces the lignin-comprising biomass,e.g., the lignin in the wood, wood pulp, Kraft pulp, paper, paperproduct or paper pulp.

The invention provides methods for treating a lignin-comprising biomass,e.g., a wood, a wood pulp, a Kraft pulp, a paper product, a paper or apaper pulp under high temperature and basic pH conditions, the methodcomprising: (a) providing at least one polypeptide having a xylanase, amannanase and/or a glucanase activity, wherein the polypeptide retainsxylanase, a mannanase and/or a glucanase activity under conditionscomprising a temperature of at least about 80° C., 85° C., 90° C. ormore, and a basic pH of at least about pH 10.5, pH 11, pH 12, pH 12.5 ormore (basic), wherein the polypeptide comprises a xylanase, a mannanaseand/or a glucanase having a sequence of the invention, or the xylanase,a mannanase and/or a glucanase is encoded by a nucleic acid comprising asequence of the invention, or an enzymatically active fragment thereof(b) providing a lignin-comprising biomass, e.g., a wood, a wood pulp, aKraft pulp, a paper, a paper product or a paper pulp; and (c) contactingthe wood, wood pulp, Kraft pulp, paper, paper product or paper pulp withthe polypeptide of step (a) under conditions comprising a temperature ofat least about 80° C., 85° C., 90° C. or more, and a basic pH of atleast about pH 10.5, pH 11, pH 12, pH 12.5 or more (basic), wherein thepolypeptide catalyzes hydrolysis of compounds in the lignin-comprisingbiomass, e.g., wood, wood pulp, Kraft pulp, paper, paper product orpaper pulp, and wherein in one aspect (optionally) the wood, wood pulp,Kraft pulp, paper, paper product or paper pulp comprises a softwood andhardwood, or the wood, wood pulp, Kraft pulp, paper or paper pulp isderived from a softwood and hardwood; and wherein in one aspect(optionally) after the treatment the pulp has a consistency of at leastabout 10%, or at least about 32%.

The invention provides methods for decoloring a biomass, e.g., a wood, awood pulp, a Kraft pulp, a paper, a paper product or a paper pulp underhigh temperature and basic pH conditions, the method comprising: (a)providing at least one polypeptide having a xylanase, a mannanase and/ora glucanase activity, wherein the polypeptide retains xylanase, amannanase and/or a glucanase activity under conditions comprising atemperature of at least about 80° C., 85° C., 90° C. or more, and abasic pH of at least about pH 10.5, pH 11, pH 12, pH 12.5 or more(basic), wherein the polypeptide comprises a xylanase, a mannanaseand/or a glucanase having a sequence of the invention, or the xylanase,a mannanase and/or a glucanase is encoded by a nucleic acid comprising asequence of the invention, or an enzymatically active fragment thereof;(b) providing a biomass, e.g., a wood, a wood pulp, a Kraft pulp, apaper, a paper product or a paper pulp; and (c) contacting the wood,wood pulp, Kraft pulp, paper, paper product or paper pulp with thepolypeptide of step (a) under conditions comprising a temperature of atleast about 80° C., 85° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95°C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C.,103.5° C., 104° C., 105° C., 107° C., 108° C., 109° C. or 110° C., ormore, and a basic pH of at least about pH 9.5, pH 10.0, pH 10.5, pH 11,pH 12, pH 12.5 or more (basic), wherein the polypeptide catalyzeshydrolysis of compounds in the biomass, e.g., a wood, wood pulp, Kraftpulp, paper, paper product or paper pulp, thereby decoloring (e.g.,bleaching) the biomass, e.g., a wood, wood pulp, Kraft pulp, paper,paper product or paper pulp.

The invention provides methods for reducing the use of decoloring (e.g.,bleaching) chemicals in a biomass, e.g., a wood, a wood pulp, a Kraftpulp, a paper, a paper product or a paper pulp decoloring (e.g.,bleaching) process under high temperature and basic pH conditions, themethod comprising: (a) providing at least one polypeptide having axylanase, a mannanase and/or a glucanase activity, wherein thepolypeptide retains xylanase, a mannanase and/or a glucanase activityunder conditions comprising a temperature of at least about 80° C., 85°C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98°C., 99° C., 100° C., 101° C., 102° C., 103° C., 103.5° C., 104° C., 105°C., 107° C., 108° C., 109° C. or 110° C., or more, and a basic pH of atleast about pH 10.5, pH 11, pH 12, pH 12.5 or more (basic), wherein thepolypeptide comprises a xylanase, a mannanase and/or a glucanase havinga sequence of the invention, or the xylanase, a mannanase and/or aglucanase is encoded by a nucleic acid comprising a sequence of theinvention, or an enzymatically active fragment thereof; (b) providing abiomass, e.g., a wood, a wood pulp, a Kraft pulp, a paper, a paperproduct or a paper pulp; and (c) contacting the wood, wood pulp, Kraftpulp, paper, paper product or paper pulp with the polypeptide of step(a) under conditions comprising a temperature of at least about 80° C.,85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C.,94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102°C., 103° C., 103.5° C., 104° C., 105° C., 107° C., 108° C., 109° C. or110° C., or more, and a basic pH of at least about pH 10.5, pH 11, pH12, pH 12.5 or more (basic), wherein the polypeptide catalyzeshydrolysis of compounds in the biomass, e.g., wood, wood pulp, Kraftpulp, paper, paper product or paper pulp, thereby biobleaching thebiomass, e.g., wood, wood pulp, Kraft pulp, paper, paper product orpaper pulp and reducing the use of decoloring (e.g., bleaching)chemicals in the decoloring (e.g., bleaching) process; wherein in oneaspect (optionally) the decoloring (e.g., bleaching) chemical comprisesa chlorine, a chlorine dioxide, a caustic, a peroxide, or anycombination thereof.

The invention provides methods for paper or pulp deinking under hightemperature and basic pH conditions, the method comprising: (a)providing at least one polypeptide having a xylanase, a mannanase and/ora glucanase activity, wherein the polypeptide retains xylanase, amannanase and/or a glucanase activity under conditions comprising atemperature of at least about 80° C., 85° C., 86° C., 87° C., 88° C.,89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C.,98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 103.5° C., 104° C.,105° C., 107° C., 108° C., 109° C. or 110° C., or more, and a basic pHof at least about pH 11, wherein the polypeptide comprises a xylanase, amannanase and/or a glucanase having a sequence of the invention, or thexylanase, a mannanase and/or a glucanase is encoded by a nucleic acidcomprising a sequence of the invention, or an enzymatically activefragment thereof; (b) providing an ink-comprising biomass, e.g., a wood,wood pulp, Kraft pulp, paper, a paper product or paper pulp; and (c)contacting the biomass, e.g., wood, wood pulp, Kraft pulp, paper, paperproduct or paper pulp with the polypeptide of step (a) under conditionscomprising a temperature of at least about 85° C. and a basic pH of atleast about pH 11,

wherein the polypeptide catalyzes hydrolysis of compounds in thebiomass, e.g., wood, wood pulp, Kraft pulp, paper or paper pulp, therebyfacilitating deinking of the biomass, e.g., wood, wood pulp, Kraft pulp,paper, paper product or paper pulp.

The invention provides methods for releasing a lignin from a biomass,e.g., a wood, a wood pulp, a Kraft pulp, a paper, a paper product or apaper pulp under high temperature and basic pH conditions, the methodcomprising: (a) providing at least one polypeptide having a xylanase, amannanase and/or a glucanase activity, wherein the polypeptide retainsxylanase, a mannanase and/or a glucanase activity under conditionscomprising a temperature of at least about 80° C., 85° C., 86° C., 87°C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96°C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 103.5°C., 104° C., 105° C., 107° C., 108° C., 109° C. or 110° C., or more, anda basic pH of at least about pH 11, wherein the polypeptide comprises axylanase, a mannanase and/or a glucanase having a sequence of theinvention, or the xylanase, a mannanase and/or a glucanase is encoded bya nucleic acid comprising a sequence of the invention, or anenzymatically active fragment thereof; (b) providing a lignin-comprisingbiomass, e.g., wood, wood pulp, Kraft pulp, paper, paper product orpaper pulp; and (c) contacting the wood, wood pulp, Kraft pulp, paper,paper product or a paper pulp of step (b) with the polypeptide of step(a) under conditions comprising a temperature of at least about 80° C.,85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C.,94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102°C., 103° C., 103.5° C., 104° C., 105° C., 107° C., 108° C., 109° C. or110° C., or more, and a basic pH of at least about pH 11, wherein thepolypeptide catalyzes hydrolysis of compounds in the wood, wood pulp,Kraft pulp, paper, paper product or paper pulp, thereby facilitatingrelease of lignin from the biomass, e.g., wood, wood pulp, Kraft pulp,paper, paper product or paper pulp; wherein in one aspect (optionally)after the treatment the pulp has a consistency of about 10%.

The invention provides compositions comprising a biomass, e.g., wood, awood pulp, a Kraft pulp, a paper, a paper product or a paper pulpcomprising a polypeptide having a xylanase, a mannanase and/or aglucanase activity, wherein the polypeptide has a sequence of theinvention, or the polypeptide is encoded by a nucleic acid comprising asequence of the invention, or an enzymatically active fragment thereof,wherein in one aspect (e.g., optionally) the biomass, e.g., wood, woodpulp, Kraft pulp, paper, paper product or paper pulp comprises asoftwood and hardwood, or the wood, wood pulp, Kraft pulp, paper, paperproduct or paper pulp derived from a softwood and hardwood.

The invention provides methods for making ethanol comprising contactinga starch-comprising composition with a polypeptide having a xylanase, amannanase and/or a glucanase activity, wherein the polypeptide has asequence of the invention, or the polypeptide is encoded by a nucleicacid comprising a sequence of the invention, or an enzymatically activefragment thereof. The invention provides compositions compositionscomprising an ethanol and a polypeptide having a xylanase, a mannanaseand/or a glucanase activity, wherein the polypeptide has a sequence ofthe invention, or the polypeptide is encoded by a nucleic acidcomprising a sequence of the invention, or an enzymatically activefragment thereof. The invention provides methods for making ethanolunder high temperature and basic pH conditions, the method comprising:(a) providing at least one polypeptide having a xylanase, a mannanaseand/or a glucanase activity, wherein the polypeptide retains xylanase, amannanase and/or a glucanase activity under conditions comprising atemperature of at least about 80° C., 85° C., 86° C., 87° C., 88° C.,89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C.,98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 103.5° C., 104° C.,105° C., 107° C., 108° C., 109° C. or 110° C., or more, and a basic pHof at least about pH 11, wherein the polypeptide comprises a xylanase, amannanase and/or a glucanase having a sequence of the invention, or thexylanase, a mannanase and/or a glucanase is encoded by a nucleic acidcomprising a sequence of the invention, or an enzymatically activefragment thereof; (b) providing a starch-comprising compositioncomprising a wood, wood pulp, Kraft pulp, paper, a paper product orpaper pulp; and (c) contacting the composition of step (b) with thepolypeptide of step (a) under conditions comprising a temperature of atleast about 80° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90° C., 91°C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100°C., 101° C., 102° C., 103° C., 103.5° C., 104° C., 105° C., 107° C.,108° C., 109° C. or 110° C., or more, and a basic pH of at least aboutpH 11, wherein the polypeptide catalyzes hydrolysis of compounds in thewood, wood pulp, Kraft pulp, paper or paper pulp, thereby generatingethanol from the wood, wood pulp, Kraft pulp, paper, paper product orpaper pulp.

The invention provides pharmaceutical compositions comprising apolypeptide having a xylanase, a mannanase and/or a glucanase activity,wherein the polypeptide comprises a sequence of the invention, or thepolypeptide is encoded by a nucleic acid comprising a sequence of theinvention, or an enzymatically active fragment thereof. In one aspect,the invention provides methods for eliminating or protecting animalsfrom a microorganism comprising a xylan comprising administering apolypeptide of the invention. The microorganism can be a bacteriumcomprising a xylan, e.g., Salmonella.

In one aspect, the pharmaceutical composition acts as a digestive aid oran anti-microbial (e.g., against Salmonella). In one aspect, thetreatment is prophylactic. In one aspect, the invention provides oralcare products comprising a polypeptide of the invention having axylanase, a mannanase and/or a glucanase activity, or a xylanase, amannanase and/or a glucanase encoded by a nucleic acid of the invention.The oral care product can comprise a toothpaste, a dental cream, a gelor a tooth powder, an odontic, a mouth wash, a pre- or post brushingrinse formulation, a chewing gum, a lozenge or a candy. The inventionprovides contact lens cleaning compositions comprising a polypeptide ofthe invention having a xylanase, a mannanase and/or a glucanaseactivity, or a xylanase, a mannanase and/or a glucanase encoded by anucleic acid of the invention.

The invention provides chimeric glycosidases, xylanases and/orglucanases comprising a polypeptide (e.g., xylanase, a mannanase and/ora glucanase) sequence of the invention and at least one heterologouscarbohydrate-binding module (CBM), wherein in one aspect (optionally)the CBM comprises a CBM3a, CBM3b, CBM4, CBM6, CBM22 or X14, or a CBM aslisted and discussed, below. The invention also provides chimericglycosidases, xylanases and/or glucanases comprising at least oneheterologous carbohydrate-binding module (CBM), wherein the CBMcomprises a carbohydrate-binding subsequence of a xylanase sequence ofthe invention, or a carbohydrate-binding subsequence comprising a X14.The invention provides methods for designing a chimeric glycosidase,xylanase, a mannanase and/or a glucanase having a newcarbohydrate-binding specificity or an enhanced carbohydrate-bindingspecificity, comprising inserting a heterologous or an additionalendogenous carbohydrate-binding module (CBM) into a glycosidases,xylanases and/or glucanases, wherein the CBM comprises acarbohydrate-binding subsequence of a glycosidase, xylanase, mannanaseand/or glucanase sequence of the invention, or a carbohydrate-bindingsubsequence comprising a X14, or alternatively a heterologous CBM, or anadditional endogenous CBM, is inserted into a xylanase, a mannanaseand/or a glucanase sequence of the invention.

The invention provides enzyme mixtures, or “cocktails” comprising atleast one enzyme of the invention and one or more other enzyme(s), whichcan be another xylanase, mannanase and/or glucanase, or any otherenzyme; for example, the “cocktails” of the invention, in addition to atleast one enzyme of this invention, can comprise any other enzyme, suchas xylanase (not of this invention), cellulases, lipases, esterases,proteases, or endoglycosidases, endo-beta.-1,4-glucanases,beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases,catalases, laccases, amylases, glucoamylases, pectinases, reductases,oxidases, phenoloxidases, ligninases, pullulanases, arabinanases,hemicellulases, mannanases, xyloglucanases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases, to namejust a few examples. In alternative embodiments, these enzyme mixtures,or “cocktails” comprising at least one enzyme of the invention can beused in any process or method of the invention, or composition of theinvention, e.g., in foods or feeds, food or feed supplements, textiles,papers, processed woods, etc. and methods for making them, and incompositions and methods for treating paper, pulp, wood, paper, pulp orwood waste or by-products, and the like, and in the final productsthereof.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of aspects of the invention andare not meant to limit the scope of the invention as encompassed by theclaims.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

FIG. 5 is a schematic flow diagram of an exemplary routine screeningprotocol to determine whether a xylanase of the invention is useful inpretreating paper pulp, as described in detail in Example 3, below.

FIG. 6 illustrates a biobleaching industrial process of the invention,as described in detail in Example 5, below.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides glycosyl hydrolases, including xylanases (e.g.,endoxylanases) and/or a glucanases, and polynucleotides encoding themand methods of making and using them. Glycosyl hydrolase, includingxylanase, mannanase and/or glucanase activity, of the polypeptides ofthe invention encompasses enzymes having hydrolase activity, forexample, enzymes capable of hydrolyzing glycosidic linkages in apolysaccharide, for example a glycosidic linkage present in xylan, e.g.,catalyzing hydrolysis of internal β-1,4-xylosidic linkages. Thexylanases and/or a glucanases of the invention can be used to makeand/or process foods, feeds, nutritional supplements, textiles,detergents and the like. The xylanases and/or a glucanases of theinvention can be used in pharmaceutical compositions and dietary aids.

Xylanases and/or a glucanases of the invention are particularly usefulin baking, animal feed, beverage and wood, wood pulp, Kraft pulp, paper,paper product or paper pulp processes. In one aspect, an enzyme of theinvention is thermotolerant and/or tolerant of high and/or low pHconditions. For example, in one aspect, a xylanase, a mannanase and/or aglucanase of the invention retains activity under conditions comprisinga temperature of at least about 80° C., 85° C., 86° C., 87° C., 88° C.,89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C., 97° C.,98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 103.5° C., 104° C.,105° C., 107° C., 108° C., 109° C. or 110° C., or more, and a basic pHof at least about pH 11, or more.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid encoding at least one polypeptide having axylanase (e.g., an endoxylanase), a mannanase and/or a glucanaseactivity, or other activity as described herein, wherein the nucleicacid comprises a sequence having at least about 50% to 99%, or more, orcomplete (100%) sequence identity (homology) to SEQ ID NO:1 having oneor more nucleotide residue changes (modifications, mutations) as setforth in Table 1, and as described herein, over a region of betweenabout 10 to 2500, or more residues, or the full length of a cDNA,transcript (mRNA) or gene. Nucleic acids of the invention includes thoseencoding a polypeptide of this invention, which includes, e.g., SEQ IDNO:2 having one or more amino acid residue changes (mutations) as setforth in Table 1 and as described herein, and also including a genus ofpolypeptides having various sequence identities based on the exemplarySEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22 or SEQ ID NO:24.

TABLE 1 Other Codons Clone Name for same Average Aver- (Xyl 11 and XylOriginal Changed AA original changed Average WT- age % 11 mutants)Residue Codon To change AA to AA A560 A560 of WT Xyl 11 WT 100 100Xyl 11 mutant 1 108 TTC AAG AAA F K 0.655 0.740 87.5 Xyl 11 mutant 2 21TTC TAC TAT F Y 0.815 0.813 100.3 Xyl 11 mutant 3 189 TCC CAG CAA S Q0.909 0.906 100.4 Xyl 11 mutant 4 150 GTA GCC GCT, V A 0.634 0.623 101.8GCA, GCG Xyl 11 mutant 5 9 CCC GAC GAT P D 0.570 0.556 102.5Xyl 11 mutant 6 188 AGC GAG GAA S E 0.749 0.722 104.1 Xyl 11 mutant 7125 CAG TAC TAT Q Y 0.936 0.902 104.2 Xyl 11 mutant 8 73 GGC GTC GTT, GV 0.769 0.736 104.6 GTA, GTG Xyl 11 mutant 9 73 GGC GAG GAA G E 0.9650.902 106.6 Xyl 11 mutant 10 33 CTG GCG GCT, L A 0.795 0.736 108.0 GCC,GCA Xyl 11 mutant 11  38 CGT CAC CAT R H 0.969 0.894 108.3Xyl 11 mutant 12 17 TTC GTC GTT, F V 0.981 0.901 109.0 GTA, GTGXyl 11 mutant 13 63 ATC GTC GTT, I V 0.996 0.901 110.5 GTA, GTGXyl 11 mutant 14 44 AGC ACG ACT, S T 1.008 0.894 112.7 ACC, ACAXyl 11 mutant 15 73 GGC TAC TAT G Y 0.958 0.813 118.2 Xyl 11 mutant 16 4ACC CAC CAT T H 1.057 0.881 119.9 Xyl 11 mutant 17 4 ACC CGC CGT, T R1.087 0.906 120.1 CGA, CGG, AGA, AGG Xyl 11 mutant 18 4 ACC AAC AAT T N1.092 0.881 123.7

The invention provides variants of polynucleotides or polypeptides ofthe invention, which comprise sequences modified at one or more basepairs, codons, introns, exons, or amino acid residues (respectively) yetstill retain the biological activity of a xylanase, a mannanase and/or aglucanase of the invention. Variants can be produced by any number ofmeans included methods such as, for example, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (e.g., GeneReassembly, see, e.g., U.S. Pat.No. 6,537,776), GS SM and any combination thereof.

The term “saturation mutagenesis”, “gene site saturation mutagenesis” or“GSSM” includes a method that uses degenerate oligonucleotide primers tointroduce point mutations into a polynucleotide, as described in detail,below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids (e.g., nucleic acids encodingpolypeptides having glycosyl hydrolase activity, e.g., a xylanase, amannanase and/or a glucanase activity; including enzymes having at leastone sequence modification of an exemplary nucleic acid sequence of theinvention (as defined above), wherein the sequence modificationcomprises one or more nucleotide residue changes (or the equivalentthereof) as set forth in Table 1, or at least one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, seventeen or eighteen, or some or all of the followingnucleotide residue changes: the codon encoding amino acid residue 4changed from ACC to AAC; the codon encoding amino acid residue 4 changedfrom ACC to CGC; the codon encoding amino acid residue 4 changed fromACC to CAC; the codon encoding amino acid residue 9 changed from CCC toGAC; the codon encoding amino acid residue 17 changed from TTC to GTC;the codon encoding amino acid residue 21 changed from TTC to TAC; thecodon encoding amino acid residue 33 changed from CTG to GCG; the codonencoding amino acid residue 38 changed from CGT to CAC; the codonencoding amino acid residue 44 changed from AGC to ACG; the codonencoding amino acid residue 63 changed from ATC to GTC; the codonencoding amino acid residue 73 changed from GGC to TAC; the codonencoding amino acid residue 73 changed from GGC to GAG; the codonencoding amino acid residue 73 changed from GGC to GTC; the codonencoding amino acid residue 108 changed from TTC to AAG; the codonencoding amino acid residue 125 changed from CAG to TAC; the codonencoding amino acid residue 150 changed from GTA to GCC; the codonencoding amino acid residue 188 changed from AGC to GAG; and/or, thecodon encoding amino acid residue 189 changed from TCC to CAG, includingexpression cassettes such as expression vectors, encoding thepolypeptides of the invention.

The invention also includes methods for discovering new xylanase,mannanase and/or glucanase sequences using the nucleic acids of theinvention. The invention also includes methods for inhibiting theexpression of xylanase, mannanase and/or glucanase genes, transcriptsand polypeptides using the nucleic acids of the invention. Also providedare methods for modifying the nucleic acids of the invention by, e.g.,synthetic ligation reassembly, optimized directed evolution systemand/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like.

In one aspect, the invention also provides xylanase- and/orglucanase-encoding nucleic acids with a common novelty in that they arederived from an environmental source, or a bacterial source, or anarchaeal source.

In practicing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

One aspect of the invention is an isolated nucleic acid comprising oneof the sequences of The invention and sequences substantially identicalthereto, the sequences complementary thereto, or a fragment comprisingat least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or500 consecutive bases of one of the sequences of a Sequence of theinvention (or the sequences complementary thereto). The isolated,nucleic acids may comprise DNA, including cDNA, genomic DNA andsynthetic DNA. The DNA may be double-stranded or single-stranded and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. Alternatively, the isolated nucleic acids may comprise RNA.

Accordingly, another aspect of the invention is an isolated nucleic acidwhich encodes one of the polypeptides of the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids of one of the polypeptides of the invention. Thecoding sequences of these nucleic acids may be identical to one of thecoding sequences of one of the nucleic acids of the invention, or afragment thereof or may be different coding sequences which encode oneof the polypeptides of the invention, sequences substantially identicalthereto and fragments having at least 5, 10, 15, 20, 25, 30, 35, 40, 50,75, 100, or 150 consecutive amino acids of one of the polypeptides ofthe invention, as a result of the redundancy or degeneracy of thegenetic code. The genetic code is well known to those of skill in theart and can be obtained, for example, on page 214 of B. Lewin, Genes VI,Oxford University Press, 1997.

The isolated nucleic acid which encodes one of the polypeptides of theinvention and sequences substantially identical thereto, may include,but is not limited to: only the coding sequence of a nucleic acid of theinvention and sequences substantially identical thereto and additionalcoding sequences, such as leader sequences or proprotein sequences andnon-coding sequences, such as introns or non-coding sequences 5′ and/or3′ of the coding sequence. Thus, as used herein, the term“polynucleotide encoding a polypeptide” encompasses a polynucleotidewhich includes only the coding sequence for the polypeptide as well as apolynucleotide which includes additional coding and/or non-codingsequence.

Alternatively, the nucleic acid sequences of the invention and sequencessubstantially identical thereto, may be mutagenized using conventionaltechniques, such as site directed mutagenesis, or other techniquesfamiliar to those skilled in the art, to introduce silent changes intothe polynucleotides of the invention and sequences substantiallyidentical thereto. As used herein, “silent changes” include, forexample, changes which do not alter the amino acid sequence encoded bythe polynucleotide. Such changes may be desirable in order to increasethe level of the polypeptide produced by host cells containing a vectorencoding the polypeptide by introducing codons or codon pairs whichoccur frequently in the host organism.

The invention also relates to polynucleotides which have nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptides of the invention andsequences substantially identical thereto. Such nucleotide changes maybe introduced using techniques such as site directed mutagenesis, randomchemical mutagenesis, exonuclease III deletion and other recombinant DNAtechniques. Alternatively, such nucleotide changes may be naturallyoccurring allelic variants which are isolated by identifying nucleicacids which specifically hybridize to probes comprising at least 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutivebases of one of the sequences of The invention and sequencessubstantially identical thereto (or the sequences complementary thereto)under conditions of high, moderate, or low stringency as providedherein.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (e.g., glycosyl hydrolases of the invention)generated from these nucleic acids can be individually isolated orcloned and tested for a desired activity. Any recombinant expressionsystem can be used, including bacterial, mammalian, yeast, insect orplant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS 1N MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent asense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin. Thephrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double strandediRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156. “Oligonucleotide” includeseither a single stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands that may be chemically synthesized. Suchsynthetic oligonucleotides have no 5′ phosphate and thus will not ligateto another oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide can ligate to afragment that has not been dephosphorylated.

A “coding sequence of” or a “nucleotide sequence encoding” a particularpolypeptide or protein, is a nucleic acid sequence which is transcribedand translated into a polypeptide or protein when placed under thecontrol of appropriate regulatory sequences.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as, where applicable,intervening sequences (introns) between individual coding segments(exons). “Operably linked” as used herein refers to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments.Typically, it refers to the functional relationship of transcriptionalregulatory sequence to a transcribed sequence. For example, a promoteris operably linked to a coding sequence, such as a nucleic acid of theinvention, if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as a xylanase, mannanase and/orglucanase of the invention) in a host compatible with such sequences.Expression cassettes include at least a promoter operably linked withthe polypeptide coding sequence; and, in one aspect, with othersequences, e.g., transcription termination signals. Additional factorsnecessary or helpful in effecting expression may also be used, e.g.,enhancers. Thus, expression cassettes also include plasmids, expressionvectors, recombinant viruses, any form of recombinant “naked DNA”vector, and the like. A “vector” comprises a nucleic acid that caninfect, transfect, transiently or permanently transduce a cell. It willbe recognized that a vector can be a naked nucleic acid, or a nucleicacid complexed with protein or lipid. The vector in one aspect comprisesviral or bacterial nucleic acids and/or proteins, and/or membranes(e.g., a cell membrane, a viral lipid envelope, etc.). Vectors include,but are not limited to replicons (e.g., RNA replicons, bacteriophages)to which fragments of DNA may be attached and become replicated. Vectorsthus include, but are not limited to RNA, autonomous self-replicatingcircular or linear DNA or RNA (e.g., plasmids, viruses, and the like,see, e.g., U.S. Pat. No. 5,217,879), and include both the expression andnon-expression plasmids. Where a recombinant microorganism or cellculture is described as hosting an “expression vector” this includesboth extra-chromosomal circular and linear DNA and DNA that has beenincorporated into the host chromosome(s). Where a vector is beingmaintained by a host cell, the vector may either be stably replicated bythe cells during mitosis as an autonomous structure, or is incorporatedwithin the host's genome.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors that ensure that genes encoding proteins specific to agiven tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

As used herein, the term “isolated” means that the material (e.g., anucleic acid, a polypeptide, a cell) is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition and still be isolated inthat such vector or composition is not part of its natural environment.As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library have been conventionally purified toelectrophoretic homogeneity. The sequences obtained from these clonescould not be obtained directly either from the library or from totalhuman DNA. The purified nucleic acids of the invention have beenpurified from the remainder of the genomic DNA in the organism by atleast 10⁴-10⁶ fold. However, the term “purified” also includes nucleicacids that have been purified from the remainder of the genomic DNA orfrom other sequences in a library or other environment by at least oneorder of magnitude, typically two or three orders and more typicallyfour or five orders of magnitude.

As used herein, the term “recombinant” means that the nucleic acid isadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. Additionally, to be “enriched” the nucleic acidswill represent 5% or more of the number of nucleic acid inserts in apopulation of nucleic acid backbone molecules. Backbone moleculesaccording to the invention include nucleic acids such as expressionvectors, self-replicating nucleic acids, viruses, integrating nucleicacids and other vectors or nucleic acids used to maintain or manipulatea nucleic acid insert of interest. Typically, the enriched nucleic acidsrepresent 15% or more of the number of nucleic acid inserts in thepopulation of recombinant backbone molecules. More typically, theenriched nucleic acids represent 50% or more of the number of nucleicacid inserts in the population of recombinant backbone molecules. In aone aspect, the enriched nucleic acids represent 90% or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules.

“Plasmids” are designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed herein are known in the art and will be apparent to theordinarily skilled artisan. “Plasmids” can be commercially available,publicly available on an unrestricted basis, or can be constructed fromavailable plasmids in accord with published procedures. Equivalentplasmids to those described herein are known in the art and will beapparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion, gel electrophoresis may beperformed to isolate the desired fragment.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature and arewell known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature. In alternativeaspects, nucleic acids of the invention are defined by their ability tohybridize under various stringency conditions (e.g., high, medium, andlow), as set forth herein.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDSand 200 ug/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lad, lacZ, T3, T7, gpt, lambda P_(R), PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.A promoter sequence is “operably linked to” a coding sequence when RNApolymerase which initiates transcription at the promoter will transcribethe coding sequence into mRNA.Promoters suitable for expressing apolypeptide in bacteria include the E. coli lac or trp promoters, thelad promoter, the lacZ promoter, the T3 promoter, the T7 promoter, thegpt promoter, the lambda PR promoter, the lambda PL promoter, promotersfrom operons encoding glycolytic enzymes such as 3-phosphoglyceratekinase (PGK), and the acid phosphatase promoter. Eukaryotic promotersinclude the CMV immediate early promoter, the HSV thymidine kinasepromoter, heat shock promoters, the early and late SV40 promoter, LTRsfrom retroviruses, and the mouse metallothionein-I promoter. Otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses may also be used. Promoters suitablefor expressing the polypeptide or fragment thereof in bacteria includethe E. coli lac or trp promoters, the lac/promoter, the lacZ promoter,the T3 promoter, the T7 promoter, the gpt promoter, the lambda P_(R)promoter, the lambda P_(L) promoter, promoters from operons encodingglycolytic enzymes such as 3-phosphoglycerate kinase (PGK) and the acidphosphatase promoter. Fungal promoters include the V factor promoter.Eukaryotic promoters include the CMV immediate early promoter, the HSVthymidine kinase promoter, heat shock promoters, the early and late SV40promoter, LTRs from retroviruses and the mouse metallothionein-Ipromoter. Other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses may also be used.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express a xylanase, mannanaseand/or glucanase of the invention in a tissue-specific manner. Theinvention also provides plants or seeds that express a xylanase,mannanase and/or glucanase of the invention in a tissue-specific manner.The tissue-specificity can be seed specific, stem specific, leafspecific, root specific, fruit specific and the like.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol. 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassaya vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression of xylanase-and/or glucanase-expressing nucleic acid in a specific tissue, organ orcell type (i.e. tissue-specific promoters) or may be otherwise undermore precise environmental or developmental control or under the controlof an inducible promoter. Examples of environmental conditions that mayaffect transcription include anaerobic conditions, elevated temperature,the presence of light, or sprayed with chemicals/hormones. For example,the invention incorporates the drought-inducible promoter of maize (Busk(1997) supra); the cold, drought, and high salt inducible promoter frompotato (Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene API; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fb12A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements El promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant. MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically-(e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the xylanase- and/or glucanase-producing nucleic acids ofthe invention will allow the grower to select plants with the optimalxylanase, mannanase and/or glucanase expression and/or activity. Thedevelopment of plant parts can thus controlled. In this way theinvention provides the means to facilitate the harvesting of plants andplant parts. For example, in various embodiments, the maize In2-2promoter, activated by benzenesulfonamide herbicide safeners, is used(De Veylder (1997) Plant Cell Physiol. 38:568-577); application ofdifferent herbicide safeners induces distinct gene expression patterns,including expression in the root, hydathodes, and the shoot apicalmeristem. Coding sequences of the invention are also under the controlof a tetracycline-inducible promoter, e.g., as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324).

In some aspects, proper polypeptide expression may requirepolyadenylation region at the 3′-end of the coding region. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant (or animal or other) genes, or from genes in theAgrobacterial T-DNA.

The term “plant” (e.g., as in a transgenic plant or plant seed of thisinvention, or plant promoter used in a vector of the invention) includeswhole plants, plant parts (e.g., leaves, stems, flowers, roots, etc.),plant protoplasts, seeds and plant cells and progeny of same; theclasses of plants that can be used to practice this invention (includingcompositions and methods) can be as broad as the class of higher plants,including plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), as well asgymnosperms; also including plants of a variety of ploidy levels,including polyploid, diploid, haploid and hemizygous states. As usedherein, the term “transgenic plant” includes plants or plant cells intowhich a heterologous nucleic acid sequence has been inserted, e.g., thenucleic acids and various recombinant constructs (e.g., expressioncassettes, such a vectors) of the invention. Transgenic plants of theinvention are also discussed, below.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding thexylanases and/or glucanases of the invention. Expression vectors andcloning vehicles of the invention can comprise viral particles,baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterialartificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul poxvirus, pseudorabies and derivatives of SV40), P1-based artificialchromosomes, yeast plasmids, yeast artificial chromosomes, and any othervectors specific for specific hosts of interest (such as bacillus,Aspergillus and yeast). Vectors of the invention can includechromosomal, non-chromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. Exemplary vectors are include: bacterial: pQEvectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors(Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic:pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).However, any other plasmid or other vector may be used so long as theyare replicable and viable in the host. Low copy number or high copynumber vectors may be employed with the present invention.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP 1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 by in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant. Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda P_(R), P_(L), and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences and 5′flanking nontranscribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required nontranscribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 by in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin and theadenovirus enhancers.

In addition, the expression vectors typically contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli and the S. cerevisiae TRP1 gene.

In some aspects, the nucleic acid encoding one of the polypeptides ofthe invention and sequences substantially identical thereto, orfragments comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50,75, 100, or 150 consecutive amino acids thereof is assembled inappropriate phase with a leader sequence capable of directing secretionof the translated polypeptide or fragment thereof. The nucleic acid canencode a fusion polypeptide in which one of the polypeptides of theinvention and sequences substantially identical thereto, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof is fused to heterologous peptides orpolypeptides, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel et al. Current Protocols inMolecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al.,Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring HarborLaboratory Press (1989. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, N.Y., (1989).

Host Cells and Transformed Cells

The invention also provides transformed cells comprising a nucleic acidsequence of the invention, e.g., a sequence encoding a xylanase, amannanase and/or a glucanase of the invention, or a vector of theinvention. The host cell may be any of the host cells familiar to thoseskilled in the art, including prokaryotic cells, eukaryotic cells, suchas bacterial cells, fungal cells, yeast cells, mammalian cells, insectcells, or plant cells. Exemplary bacterial cells include any specieswithin the genera Escherichia, Bacillus, Streptomyces, Salmonella,Pseudomonas and Staphylococcus, including, e.g., Escherichia coli,Lactococcus lactis, Bacillus subtilis, Bacillus cereus, Salmonellatyphimurium, Pseudomonas fluorescens. Exemplary fungal cells include anyspecies of Aspergillus. Exemplary yeast cells include any species ofPichia, Saccharomyces, Schizosaccharomyces, or Schwanniomyces, includingPichia pastoris, Saccharomyces cerevisiae, or Schizosaccharomyces pombe.Exemplary insect cells include any species of Spodoptera or Drosophila,including Drosophila S2 and Spodoptera Sf9. Exemplary animal cellsinclude CHO, COS or Bowes melanoma or any mouse or human cell line. Theselection of an appropriate host is within the abilities of thoseskilled in the art. Techniques for transforming a wide variety of higherplant species are well known and described in the technical andscientific literature. See, e.g., Weising (1988) Ann. Rev. Genet.22:421-477; U.S. Pat. No. 5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets are can be used.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Host cells containing the polynucleotides of interest, e.g., nucleicacids of the invention, can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression and will be apparent to the ordinarilyskilled artisan. The clones which are identified as having the specifiedenzyme activity may then be sequenced to identify the polynucleotidesequence encoding an enzyme having the enhanced activity.

The invention provides a method for overexpressing a recombinantxylanase, mannanase and/or glucanase in a cell comprising expressing avector comprising a nucleic acid of the invention, e.g., a nucleic acidcomprising a nucleic acid sequence with at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence of Theinvention over a region of at least about 100 residues, wherein thesequence identities are determined by analysis with a sequencecomparison algorithm or by visual inspection, or, a nucleic acid thathybridizes under stringent conditions to a nucleic acid sequence of Theinvention, or a subsequence thereof. The overexpression can be effectedby any means, e.g., use of a high activity promoter, a dicistronicvector or by gene amplification of the vector.

The nucleic acids of the invention can be expressed, or overexpressed,in any in vitro or in vivo expression system. Any cell culture systemscan be employed to express, or over-express, recombinant protein,including bacterial, insect, yeast, fungal or mammalian cultures.Over-expression can be effected by appropriate choice of promoters,enhancers, vectors (e.g., use of replicon vectors, dicistronic vectors(see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun. 229:295-8),media, culture systems and the like. In one aspect, gene amplificationusing selection markers, e.g., glutamine synthetase (see, e.g., Sanders(1987) Dev. Biol. Stand. 66:55-63), in cell systems are used tooverexpress the polypeptides of the invention.

Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes EP 0659215(WO 9403612 A1) (Nevalainen et al.); Lapidot, A., Mechaly, A., Shoham,Y., “Overexpression and single-step purification of a thermostablexylanase from Bacillus stearothermophilus T-6,” J. Biotechnol. November51:259-64 (1996); Liithi, E., Jasmat, N.B., Bergquist, P. L., “Xylanasefrom the extremely thermophilic bacterium Caldocellum saccharolyticum:overexpression of the gene in Escherichia coli and characterization ofthe gene product,” Appl. Environ. Microbiol. September 56:2677-83(1990); and Sung, W. L., Luk, C. K., Zahab, D. M., Wakarchuk, W.,“Overexpression of the Bacillus subtilis and circulans xylanases inEscherichia coli,” Protein Expr. Purif. June 4:200-6 (1993), althoughthese references do not teach the inventive enzymes of the instantapplication.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells, eukaryotic cells, mammalian cells,insect cells, or plant cells. As representative examples of appropriatehosts, there may be mentioned: bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, fungal cells, such as Aspergillus, yeast such as anyspecies of Pichia, Saccharomyces, Schizosaccharomyces, Schwanniomyces,including Pichia pastoris, Saccharomyces cerevisiae, orSchizosaccharomyces pombe, insect cells such as Drosophila S2 andSpodoptera SJ9, animal cells such as CHO, COS or Bowes melanoma andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps. Various mammalian cell culture systems canalso be employed to express recombinant protein. Examples of mammalianexpression systems include the COS-7 lines of monkey kidney fibroblasts(described by Gluzman, Cell, 23:175, 1981) and other cell lines capableof expressing proteins from a compatible vector, such as the C127, 3T3,CHO, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Alternatively, the polypeptides of amino acid sequences of theinvention, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,40, 50, 75, 100, or 150 consecutive amino acids thereof can besynthetically produced by conventional peptide synthesizers. In otheraspects, fragments or portions of the polypeptides may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, the fragments may be employed as intermediates forproducing the full-length polypeptides.

Cell-free translation systems can also be employed to produce one of thepolypeptides of amino acid sequences of the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof using mRNAs transcribed from a DNAconstruct comprising a promoter operably linked to a nucleic acidencoding the polypeptide or fragment thereof. In some aspects, the DNAconstruct may be linearized prior to conducting an in vitrotranscription reaction. The transcribed mRNA is then incubated with anappropriate cell-free translation extract, such as a rabbit reticulocyteextract, to produce the desired polypeptide or fragment thereof.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding the xylanases and/or glucanases of the invention, ormodified nucleic acids of the invention, can be reproduced byamplification. Amplification can also be used to clone or modify thenucleic acids of the invention. Thus, the invention providesamplification primer sequence pairs for amplifying nucleic acids of theinvention. One of skill in the art can design amplification primersequence pairs for any part of or the full length of these sequences.

In one aspect, the invention provides a nucleic acid amplified by aprimer pair of the invention, e.g., a primer pair as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and about the first(the 5′) 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having a xylanase,mannanase and/or glucanase activity, wherein the primer pair is capableof amplifying a nucleic acid comprising a sequence of the invention, orfragments or subsequences thereof. One or each member of theamplification primer sequence pair can comprise an oligonucleotidecomprising at least about 10 to 50 consecutive bases of the sequence, orabout 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25consecutive bases of the sequence. The invention provides amplificationprimer pairs, wherein the primer pair comprises a first member having asequence as set forth by about the first (the 5′) 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of a nucleic acid of theinvention, and a second member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of the complementary strand of the first member. Theinvention provides xylanases and/or glucanases generated byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. The invention providesmethods of making a xylanase, mannanase and/or glucanase byamplification, e.g., polymerase chain reaction (PCR), using anamplification primer pair of the invention. In one aspect, theamplification primer pair amplifies a nucleic acid from a library, e.g.,a gene library, such as an environmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary nucleic acid of the invention(as defined above) over a region of at least about 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550 or more, residues. The invention provides polypeptidescomprising sequences having at least about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity to an exemplarypolypeptide of the invention. The extent of sequence identity (homology)may be determined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, refers to two or more sequences that have, e.g., atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide oramino acid residue (sequence) identity, when compared and aligned formaximum correspondence, as measured using one of the known sequencecomparison algorithms or by visual inspection. Typically, thesubstantial identity exists over a region of at least about 100 residuesand most commonly the sequences are substantially identical over atleast about 150-200 residues. In some aspects, the sequences aresubstantially identical over the entire length of the coding regions.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule and provided that the polypeptideessentially retains its functional properties. A conservative amino acidsubstitution, for example, substitutes one amino acid for another of thesame class (e.g., substitution of one hydrophobic amino acid, such asisoleucine, valine, leucine, or methionine, for another, or substitutionof one polar amino acid for another, such as substitution of argininefor lysine, glutamic acid for aspartic acid or glutamine forasparagine). One or more amino acids can be deleted, for example, from axylanase, mannanase and/or glucanase polypeptide, resulting inmodification of the structure of the polypeptide, without significantlyaltering its biological activity. For example, amino- orcarboxyl-terminal amino acids that are not required for xylanase,mannanase and/or glucanase biological activity can be removed. Modifiedpolypeptide sequences of the invention can be assayed for xylanase,mannanase and/or glucanase biological activity by any number of methods,including contacting the modified polypeptide sequence with a xylanase,mannanase and/or glucanase substrate and determining whether themodified polypeptide decreases the amount of specific substrate in theassay or increases the bioproducts of the enzymatic reaction of afunctional xylanase, mannanase and/or glucanase polypeptide with thesubstrate.

Nucleic acid sequences of the invention can comprise at least 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutivenucleotides of an exemplary sequence of the invention and sequencessubstantially identical thereto. Nucleic acid sequences of the inventioncan comprise homologous sequences and fragments of nucleic acidsequences and sequences substantially identical thereto, refer to asequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity (homology) to these sequences. Homology may bedetermined using any of the computer programs and parameters describedherein, including FASTA version 3.0t78 with the default parameters.Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences of the invention. Thehomologous sequences may be obtained using any of the proceduresdescribed herein or may result from the correction of a sequencingerror. It will be appreciated that the nucleic acid sequences of theinvention and sequences substantially identical thereto, can berepresented in the traditional single character format (See the insideback cover of Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co.,New York.) or in any other format which records the identity of thenucleotides in a sequence.

Various sequence comparison programs identified elsewhere in this patentspecification are particularly contemplated for use in this aspect ofthe invention. Protein and/or nucleic acid sequence homologies may beevaluated using any of the variety of sequence comparison algorithms andprograms known in the art. Such algorithms and programs include, but areby no means limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988;Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al.,Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., MethodsEnzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol.215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of person & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project. Atleast twenty-one other genomes have already been sequenced, including,for example, M. genitalium (Fraser et al., 1995), M. jannaschii (Bult etal., 1996), H. influenzae (Fleischmann et al., 1995), E. coli (Blattneret al., 1997) and yeast (S. cerevisiae) (Mewes et al., 1997) and D.melanogaster (Adams et al., 2000). Significant progress has also beenmade in sequencing the genomes of model organism, such as mouse, C.elegans and Arabadopsis sp. Several databases containing genomicinformation annotated with some functional information are maintained bydifferent organization and are accessible via the internet One exampleof a useful algorithm is BLAST and BLAST 2.0 algorithms, which aredescribed in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977 andAltschul et al., J. Mol. Biol. 215:403-410, 1990, respectively. Softwarefor performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul et al., supra). These initialneighborhood word hits act as seeds for initiating searches to findlonger HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3 and expectations (E) of 10 and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, more preferably less thanabout 0.01 and most preferably less than about 0.001.

In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

(1) BLASTP and BLAST3 compare an amino acid query sequence against aprotein sequence database;

(2) BLASTN compares a nucleotide query sequence against a nucleotidesequence database;

(3) BLASTX compares the six-frame conceptual translation products of aquery nucleotide sequence (both strands) against a protein sequencedatabase;

(4) TBLASTN compares a query protein sequence against a nucleotidesequence database translated in all six reading frames (both strands);and

(5) TBLASTX compares the six-frame translations of a nucleotide querysequence against the six-frame translations of a nucleotide sequencedatabase.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet etal., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins17:49-61, 1993). Less preferably, the PAM or PAM250 matrices may also beused (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices forDetecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). BLASTprograms are accessible through the U.S. National Library of Medicine.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, a nucleic acid or polypeptide sequence ofthe invention can be stored, recorded, and manipulated on any mediumwhich can be read and accessed by a computer.

Accordingly, the invention provides computers, computer systems,computer readable mediums, computer programs products and the likerecorded or stored thereon the nucleic acid and polypeptide sequences ofthe invention. As used herein, the words “recorded” and “stored” referto a process for storing information on a computer medium. A skilledartisan can readily adopt any known methods for recording information ona computer readable medium to generate manufactures comprising one ormore of the nucleic acid and/or polypeptide sequences of the invention.

The polypeptides of the invention include the exemplary sequences of theinvention, and sequences substantially identical thereto, and fragmentsof any of the preceding sequences. Substantially identical, orhomologous, polypeptide sequences refer to a polypeptide sequence havingat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary sequence of the invention,e.g., a polypeptide sequences of the invention.

Homology may be determined using any of the computer programs andparameters described herein, including FASTA version 3.0t78 with thedefault parameters or with any modified parameters. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. The polypeptidefragments comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutiveamino acids of the polypeptides of the invention and sequencessubstantially identical thereto. It will be appreciated that thepolypeptide codes of amino acid sequences of the invention and sequencessubstantially identical thereto, can be represented in the traditionalsingle character format or three letter format (See Stryer, Lubert.Biochemistry, 3rd Ed., supra) or in any other format which relates theidentity of the polypeptides in a sequence.

A nucleic acid or polypeptide sequence of the invention can be stored,recorded and manipulated on any medium which can be read and accessed bya computer. As used herein, the words “recorded” and “stored” refer to aprocess for storing information on a computer medium. A skilled artisancan readily adopt any of the presently known methods for recordinginformation on a computer readable medium to generate manufacturescomprising one or more of the nucleic acid sequences of the inventionand sequences substantially identical thereto, one or more of thepolypeptide sequences of the invention and sequences substantiallyidentical thereto. Another aspect of the invention is a computerreadable medium having recorded thereon at least 2, 5, 10, 15, or 20 ormore nucleic acid sequences of the invention and sequences substantiallyidentical thereto.

Another aspect of the invention is a computer readable medium havingrecorded thereon one or more of the nucleic acid sequences of theinvention and sequences substantially identical thereto. Another aspectof the invention is a computer readable medium having recorded thereonone or more of the polypeptide sequences of the invention and sequencessubstantially identical thereto. Another aspect of the invention is acomputer readable medium having recorded thereon at least 2, 5, 10, 15,or 20 or more of the sequences as set forth above.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM) as well as othertypes of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., interne based systems),particularly computer systems which store and manipulate the sequenceinformation described herein. One example of a computer system 100 isillustrated in block diagram form in FIG. 1. As used herein, “a computersystem” refers to the hardware components, software components and datastorage components used to analyze a nucleotide sequence of a nucleicacid sequence of the invention and sequences substantially identicalthereto, or a polypeptide sequence as set forth in the amino acidsequences of the invention. The computer system 100 typically includes aprocessor for processing, accessing and manipulating the sequence data.The processor 105 can be any well-known type of central processing unit,such as, for example, the Pentium III from Intel Corporation, or similarprocessor from Sun, Motorola, Compaq, AMD or International BusinessMachines.

Typically the computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one particular aspect, the computer system 100 includes a processor105 connected to a bus which is connected to a main memory 115(preferably implemented as RAM) and one or more internal data storagedevices 110, such as a hard drive and/or other computer readable mediahaving data recorded thereon. In some aspects, the computer system 100further includes one or more data retrieving device 118 for reading thedata stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet)etc. In some aspects, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

Software for accessing and processing the nucleotide sequences of anucleic acid sequence of the invention and sequences substantiallyidentical thereto, or a polypeptide sequence of the invention andsequences substantially identical thereto, (such as search tools,compare tools and modeling tools etc.) may reside in main memory 115during execution.

In some aspects, the computer system 100 may further comprise a sequencecomparison algorithm for comparing a nucleic acid sequence of theinvention and sequences substantially identical thereto, or apolypeptide sequence of the invention and sequences substantiallyidentical thereto, stored on a computer readable medium to a referencenucleotide or polypeptide sequence(s) stored on a computer readablemedium. A “sequence comparison algorithm” refers to one or more programswhich are implemented (locally or remotely) on the computer system 100to compare a nucleotide sequence with other nucleotide sequences and/orcompounds stored within a data storage means. For example, the sequencecomparison algorithm may compare the nucleotide sequences of a nucleicacid sequence of the invention and sequences substantially identicalthereto, or a polypeptide sequence of the invention and sequencessubstantially identical thereto, stored on a computer readable medium toreference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

FIG. 2 is a flow diagram illustrating one aspect of a process 200 forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

Accordingly, one aspect of the invention is a computer system comprisinga processor, a data storage device having stored thereon a nucleic acidsequence of the invention and sequences substantially identical thereto,or a polypeptide sequence of the invention and sequences substantiallyidentical thereto, a data storage device having retrievably storedthereon reference nucleotide sequences or polypeptide sequences to becompared to a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto and a sequencecomparer for conducting the comparison. The sequence comparer mayindicate a homology level between the sequences compared or identifystructural motifs in the above described nucleic acid code of nucleicacid sequences of the invention and sequences substantially identicalthereto, or a polypeptide sequence of the invention and sequencessubstantially identical thereto, or it may identify structural motifs insequences which are compared to these nucleic acid codes and polypeptidecodes. In some aspects, the data storage device may have stored thereonthe sequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of thenucleic acid sequences of the invention and sequences substantiallyidentical thereto, or the polypeptide sequences of the invention andsequences substantially identical thereto.

Another aspect of the invention is a method for determining the level ofhomology between a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto and a referencenucleotide sequence. The method including reading the nucleic acid codeor the polypeptide code and the reference nucleotide or polypeptidesequence through the use of a computer program which determines homologylevels and determining homology between the nucleic acid code orpolypeptide code and the reference nucleotide or polypeptide sequencewith the computer program. The computer program may be any of a numberof computer programs for determining homology levels, including thosespecifically enumerated herein, (e.g., BLAST2N with the defaultparameters or with any modified parameters). The method may beimplemented using the computer systems described above. The method mayalso be performed by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 ormore of the above described nucleic acid sequences of the invention, orthe polypeptide sequences of the invention through use of the computerprogram and determining homology between the nucleic acid codes orpolypeptide codes and reference nucleotide sequences or polypeptidesequences.

FIG. 3 is a flow diagram illustrating one aspect of a process 250 in acomputer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is preferably in the single letter amino acidcode so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are not any more characters to read, then the process 250 movesto a state 276 wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of a nucleic acid sequence as setforth in the invention, to one or more reference nucleotide sequences inorder to determine whether the nucleic acid code of a nucleic acidsequence of the invention and sequences substantially identical thereto,differs from a reference nucleic acid sequence at one or more positions.In one aspect such a program records the length and identity ofinserted, deleted or substituted nucleotides with respect to thesequence of either the reference polynucleotide or a nucleic acidsequence of the invention and sequences substantially identical thereto.In one aspect, the computer program may be a program which determineswhether a nucleic acid sequence of the invention and sequencessubstantially identical thereto, contains a single nucleotidepolymorphism (SNP) with respect to a reference nucleotide sequence.

Another aspect of the invention is a method for determining whether anucleic acid sequence of the invention and sequences substantiallyidentical thereto, differs at one or more nucleotides from a referencenucleotide sequence comprising the steps of reading the nucleic acidcode and the reference nucleotide sequence through use of a computerprogram which identifies differences between nucleic acid sequences andidentifying differences between the nucleic acid code and the referencenucleotide sequence with the computer program. In some aspects, thecomputer program is a program which identifies single nucleotidepolymorphisms. The method may be implemented by the computer systemsdescribed above and the method illustrated in FIG. 3. The method mayalso be performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 40 ormore of the nucleic acid sequences of the invention and sequencessubstantially identical thereto and the reference nucleotide sequencesthrough the use of the computer program and identifying differencesbetween the nucleic acid codes and the reference nucleotide sequenceswith the computer program.

In other aspects the computer based system may further comprise anidentifier for identifying features within a nucleic acid sequence ofthe invention or a polypeptide sequence of the invention and sequencessubstantially identical thereto.

An “identifier” refers to one or more programs which identifies certainfeatures within a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto. In one aspect,the identifier may comprise a program which identifies an open readingframe in a nucleic acid sequence of the invention and sequencessubstantially identical thereto.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group. Alternatively, the features may bestructural polypeptide motifs such as alpha helices, beta sheets, orfunctional polypeptide motifs such as enzymatic active sites,helix-turn-helix motifs or other motifs known to those skilled in theart.

Once the database of features is opened at the state 306, the process300 moves to a state 308 wherein the first feature is read from thedatabase. A comparison of the attribute of the first feature with thefirst sequence is then made at a state 310. A determination is then madeat a decision state 316 whether the attribute of the feature was foundin the first sequence. If the attribute was found, then the process 300moves to a state 318 wherein the name of the found feature is displayedto the user.

The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence. It should be noted, that if thefeature attribute is not found in the first sequence at the decisionstate 316, the process 300 moves directly to the decision state 320 inorder to determine if any more features exist in the database.

Accordingly, another aspect of the invention is a method of identifyinga feature within a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto, comprisingreading the nucleic acid code(s) or polypeptide code(s) through the useof a computer program which identifies features therein and identifyingfeatures within the nucleic acid code(s) with the computer program. Inone aspect, computer program comprises a computer program whichidentifies open reading frames. The method may be performed by reading asingle sequence or at least 2, 5, 10, 15, 20, 25, 30, or 40 of thenucleic acid sequences of the invention and sequences substantiallyidentical thereto, or the polypeptide sequences of the invention andsequences substantially identical thereto, through the use of thecomputer program and identifying features within the nucleic acid codesor polypeptide codes with the computer program.

A nucleic acid sequence of the invention and sequences substantiallyidentical thereto, or a polypeptide sequence of the invention andsequences substantially identical thereto, may be stored and manipulatedin a variety of data processor programs in a variety of formats. Forexample, a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto, may be storedas text in a word processing file, such as Microsoft WORD™ orWORDPERFECT™ or as an ASCII file in a variety of database programsfamiliar to those of skill in the art, such as DB2™, SYBASE™, orORACLE™. In addition, many computer programs and databases may be usedas sequence comparison algorithms, identifiers, or sources of referencenucleotide sequences or polypeptide sequences to be compared to anucleic acid sequence of the invention and sequences substantiallyidentical thereto, or a polypeptide sequence of the invention andsequences substantially identical thereto. The following list isintended not to limit the invention but to provide guidance to programsand databases which are useful with the nucleic acid sequences of theinvention and sequences substantially identical thereto, or thepolypeptide sequences of the invention and sequences substantiallyidentical thereto.

The programs and databases which may be used include, but are notlimited to: MacPattern (EMBL), DiscoveryBase (Molecular ApplicationsGroup), GeneMine (Molecular Applications Group), Look (MolecularApplications Group), MacLook (Molecular Applications Group), BLAST andBLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215:403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (MolecularSimulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.),HypoGen (Molecular Simulations Inc.), Insight II, (Molecular SimulationsInc.), Discover (Molecular Simulations Inc.), CHARMm (MolecularSimulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),Homology (Molecular Simulations Inc.), Modeler (Molecular SimulationsInc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design(Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.),WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), theMDL Available Chemicals Directory database, the MDL Drug Data Reportdata base, the Comprehensive Medicinal Chemistry database, Derwents'sWorld Drug Index database, the BioByteMasterFile database, the Genbankdatabase and the Genseqn database. Many other programs and data baseswould be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated, synthetic or recombinant nucleic acidsthat hybridize under stringent conditions to an exemplary sequence ofthe invention. The stringent conditions can be highly stringentconditions, medium stringent conditions and/or low stringent conditions,including the high and reduced stringency conditions described herein.In one aspect, it is the stringency of the wash conditions that setforth the conditions which determine whether a nucleic acid is withinthe scope of the invention, as discussed below.

In alternative aspects, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA (single or double stranded),antisense or sequences encoding antibody binding peptides (epitopes),motifs, active sites and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200ug/ml sheared and denatured salmon sperm DNA). In one aspect, nucleicacids of the invention are defined by their ability to hybridize underreduced stringency conditions comprising 35% formamide at a reducedtemperature of 35° C.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent) and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4−9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at T_(m)-10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

All of the foregoing hybridizations would be considered to be underconditions of high stringency.

Following hybridization, a filter can be washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content) and the nucleic acid type (e.g., RNA v. DNA). Examples ofprogressively higher stringency condition washes are as follows: 2×SSC,0.1% SDS at room temperature for 15 minutes (low stringency); 0.1×SSC,0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes. Some other examples are givenbelow.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na+ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention. Forexample, the preceding methods may be used to isolate nucleic acidshaving a sequence with at least about 97%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% homology to a nucleic acidsequence selected from the group consisting of one of the sequences ofThe invention and sequences substantially identical thereto, orfragments comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 300, 400, or 500 consecutive bases thereof and thesequences complementary thereto. Homology may be measured using thealignment algorithm. For example, the homologous polynucleotides mayhave a coding sequence which is a naturally occurring allelic variant ofone of the coding sequences described herein. Such allelic variants mayhave a substitution, deletion or addition of one or more nucleotideswhen compared to the nucleic acids of The invention or the sequencescomplementary thereto.

Additionally, the above procedures may be used to isolate nucleic acidswhich encode polypeptides having at least about 99%, 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% homology to a polypeptidehaving the sequence of one of amino acid sequences of the invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof as determined using asequence alignment algorithm (e.g., such as the FASTA version 3.0t78algorithm with the default parameters).

Oligonucleotides Probes and Methods for Using Them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with a xylanase,mannanase and/or glucanase activity or fragments thereof or foridentifying xylanase, mannanase and/or glucanase genes. In one aspect,the probe comprises at least 10 consecutive bases of a nucleic acid ofthe invention. Alternatively, a probe of the invention can be at leastabout 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of asequence as set forth in a nucleic acid of the invention. The probesidentify a nucleic acid by binding and/or hybridization. The probes canbe used in arrays of the invention, see discussion below, including,e.g., capillary arrays. The probes of the invention can also be used toisolate other nucleic acids or polypeptides.

The isolated nucleic acids of The invention and sequences substantiallyidentical thereto, the sequences complementary thereto, or a fragmentcomprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200,300, 400, or 500 consecutive bases of one of the sequences of Theinvention and sequences substantially identical thereto, or thesequences complementary thereto may also be used as probes to determinewhether a biological sample, such as a soil sample, contains an organismhaving a nucleic acid sequence of the invention or an organism fromwhich the nucleic acid was obtained. In such procedures, a biologicalsample potentially harboring the organism from which the nucleic acidwas isolated is obtained and nucleic acids are obtained from the sample.The nucleic acids are contacted with the probe under conditions whichpermit the probe to specifically hybridize to any complementarysequences from which are present therein.

Where necessary, conditions which permit the probe to specificallyhybridize to complementary sequences may be determined by placing theprobe in contact with complementary sequences from samples known tocontain the complementary sequence as well as control sequences which donot contain the complementary sequence. Hybridization conditions, suchas the salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids.

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

Many methods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures and dot blots. Protocols for each of theseprocedures are provided in Ausubel et al. Current Protocols in MolecularBiology, John Wiley 503 Sons, Inc. (1997) and Sambrook et al., MolecularCloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor LaboratoryPress (1989.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). Typically, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook,supra. Alternatively, the amplification may comprise a ligase chainreaction, 3SR, or strand displacement reaction. (See Barany, F., “TheLigase Chain Reaction in a PCR World”, PCR Methods and Applications1:5-16, 1991; E. Fahy et al., “Self-sustained Sequence Replication(3SR): An Isothermal Transcription-based Amplification SystemAlternative to PCR”, PCR Methods and Applications 1:25-33, 1991; andWalker G. T. et al., “Strand Displacement Amplification-an Isothermal invitro DNA Amplification Technique”, Nucleic Acid Research 20:1691-1696,1992). In such procedures, the nucleic acids in the sample are contactedwith the probes, the amplification reaction is performed and anyresulting amplification product is detected. The amplification productmay be detected by performing gel electrophoresis on the reactionproducts and staining the gel with an intercalator such as ethidiumbromide. Alternatively, one or more of the probes may be labeled with aradioactive isotope and the presence of a radioactive amplificationproduct may be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the ends of the sequences of Theinvention and sequences substantially identical thereto, may also beused in chromosome walking procedures to identify clones containinggenomic sequences located adjacent to the sequences of The invention andsequences substantially identical thereto. Such methods allow theisolation of genes which encode additional proteins from the hostorganism.

The isolated nucleic acids of the invention and sequences substantiallyidentical thereto, the sequences complementary thereto, or a fragmentcomprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200,300, 400, or 500 consecutive bases of one of the sequences of theinvention and sequences substantially identical thereto, or thesequences complementary thereto may be used as probes to identify andisolate related nucleic acids. In some aspects, the related nucleicacids may be cDNAs or genomic DNAs from organisms other than the onefrom which the nucleic acid was isolated. For example, the otherorganisms may be related organisms. In such procedures, a nucleic acidsample is contacted with the probe under conditions which permit theprobe to specifically hybridize to related sequences. Hybridization ofthe probe to nucleic acids from the related organism is then detectedusing any of the methods described above.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas:

For probes between 14 and 70 nucleotides in length the meltingtemperature (T_(m)) is calculated using the formula: T_(m)=81.5+16.6(log[Na+])+0.41(fraction G+C)−(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:T_(m)=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 μg/ml denatured fragmented salmon sperm DNA or 6×SSC,5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured fragmented salmonsperm DNA, 50% formamide. The formulas for SSC and Denhardt's solutionsare listed in Sambrook et al., supra.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the T_(m). Forshorter probes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the T. Typically, for hybridizations in6×SSC, the hybridization is conducted at approximately 68° C. Usually,for hybridizations in 50% formamide containing solutions, thehybridization is conducted at approximately 42° C.

Inhibiting Expression of Glycosyl Hydrolases

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acids of the invention, e.g., xylanase- and/orglucanase-encoding nucleic acids. Antisense sequences are capable ofinhibiting the transport, splicing or transcription of xylanase- and/orglucanase-encoding genes. The inhibition can be effected through thetargeting of genomic DNA or messenger RNA. The transcription or functionof targeted nucleic acid can be inhibited, for example, by hybridizationand/or cleavage. One particularly useful set of inhibitors provided bythe present invention includes oligonucleotides which are able to eitherbind xylanase, mannanase and/or glucanase gene or message, in eithercase preventing or inhibiting the production or function of xylanase,mannanase and/or glucanase. The association can be through sequencespecific hybridization. Another useful class of inhibitors includesoligonucleotides which cause inactivation or cleavage of xylanase,mannanase and/or glucanase message. The oligonucleotide can have enzymeactivity which causes such cleavage, such as ribozymes. Theoligonucleotide can be chemically modified or conjugated to an enzyme orcomposition capable of cleaving the complementary nucleic acid. A poolof many different such oligonucleotides can be screened for those withthe desired activity. Thus, the invention provides various compositionsfor the inhibition of xylanase, mannanase and/or glucanase expression ona nucleic acid and/or protein level, e.g., antisense, iRNA and ribozymescomprising xylanase, mannanase and/or glucanase sequences of theinvention and the anti-xylanase and/or anti-glucanase antibodies of theinvention.

Inhibition of xylanase, mannanase and/or glucanase expression can have avariety of industrial, medical, pharmaceutical, research, food and feedand food and feed supplement processing and other applications andprocesses. For example, inhibition of xylanase, mannanase and/orglucanase expression can slow or prevent spoilage. Spoilage can occurwhen polysaccharides, e.g., structural polysaccharides, areenzymatically degraded. This can lead to the deterioration, or rot, offruits and vegetables. In one aspect, use of compositions of theinvention that inhibit the expression and/or activity of xylanasesand/or glucanases, e.g., antibodies, antisense oligonucleotides,ribozymes and RNAi, are used to slow or prevent spoilage. Thus, in oneaspect, the invention provides methods and compositions comprisingapplication onto a plant or plant product (e.g., a cereal, a grain, afruit, seed, root, leaf, etc.) antibodies, antisense oligonucleotides,ribozymes and RNAi of the invention to slow or prevent spoilage. Thesecompositions also can be expressed by the plant (e.g., a transgenicplant) or another organism (e.g., a bacterium or other microorganismtransformed with a xylanase, mannanase and/or glucanase gene of theinvention).

The compositions of the invention for the inhibition of xylanase,mannanase and/or glucanase expression (e.g., antisense, iRNA, ribozymes,antibodies) can be used as pharmaceutical compositions, e.g., asanti-pathogen agents or in other therapies, e.g., as anti-microbialsfor, e.g., Salmonella.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of bindingxylanase, mannanase and/or glucanase message which can inhibit xylanhydrolase activity (e.g., catalyzing hydrolysis of internalβ-1,4-xylosidic linkages) by targeting mRNA. Strategies for designingantisense oligonucleotides are well described in the scientific andpatent literature, and the skilled artisan can design such xylanase,mannanase and/or glucanase oligonucleotides using the novel reagents ofthe invention. For example, gene walking/RNA mapping protocols to screenfor effective antisense oligonucleotides are well known in the art, see,e.g., Ho (2000) Methods Enzymol. 314:168-183, describing an RNA mappingassay, which is based on standard molecular techniques to provide aneasy and reliable method for potent antisense sequence selection. Seealso Smith (2000) Eur. J. Pharm. Sci. 11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl)glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisensexylanase, mannanase and/or glucanase sequences of the invention (see,e.g., Gold (1995) J. of Biol. Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding xylanase, mannanaseand/or glucanase message. These ribozymes can inhibit xylanase,mannanase and/or glucanase activity by, e.g., targeting mRNA. Strategiesfor designing ribozymes and selecting the xylanase- and/orglucanase-specific antisense sequence for targeting are well describedin the scientific and patent literature, and the skilled artisan candesign such ribozymes using the novel reagents of the invention.Ribozymes act by binding to a target RNA through the target RNA bindingportion of a ribozyme which is held in close proximity to an enzymaticportion of the RNA that cleaves the target RNA. Thus, the ribozymerecognizes and binds a target RNA through complementary base-pairing,and once bound to the correct site, acts enzymatically to cleave andinactivate the target RNA. Cleavage of a target RNA in such a mannerwill destroy its ability to direct synthesis of an encoded protein ifthe cleavage occurs in the coding sequence. After a ribozyme has boundand cleaved its RNA target, it can be released from that RNA to bind andcleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNaseP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising a xylanase, mannanase and/orglucanase enzyme sequence of the invention. The RNAi molecule cancomprise a double-stranded RNA (dsRNA) molecule, e.g., siRNA, miRNAand/or short hairpin RNA (shRNA) molecules. The RNAi molecule, e.g.,siRNA (small inhibitory RNA) can inhibit expression of a xylanase,mannanase and/or glucanase enzyme gene, and/or miRNA (micro RNA) toinhibit translation of a xylanase, mannanase and/or glucanase message.In one aspect, the RNAi molecule, e.g., siRNA and/or miRNA, is about 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or more duplex nucleotides in length. While the invention is not limitedby any particular mechanism of action, the RNAi can enter a cell andcause the degradation of a single-stranded RNA (ssRNA) of similar oridentical sequences, including endogenous mRNAs. When a cell is exposedto double-stranded RNA (dsRNA), mRNA from the homologous gene isselectively degraded by a process called RNA interference (RNAi). Apossible basic mechanism behind RNAi is the breaking of adouble-stranded RNA (dsRNA) matching a specific gene sequence into shortpieces called short interfering RNA, which trigger the degradation ofmRNA that matches its sequence. In one aspect, the RNAi's of theinvention are used in gene-silencing therapeutics, see, e.g., Shuey(2002) Drug Discov. Today 7:1040-1046. In one aspect, the inventionprovides methods to selectively degrade RNA using the RNAi's molecules,e.g., siRNA and/or miRNA, of the invention. The process may be practicedin vitro, ex vivo or in vivo. In one aspect, the RNAi molecules of theinvention can be used to generate a loss-of-function mutation in a cell,an organ or an animal.

In one aspect, intracellular introduction of the RNAi is byinternalization of a target cell specific ligand bonded to an RNAbinding protein comprising an RNAi (e.g., microRNA) is adsorbed. Theligand is specific to a unique target cell surface antigen. The ligandcan be spontaneously internalized after binding to the cell surfaceantigen. If the unique cell surface antigen is not naturallyinternalized after binding to its ligand, internalization can bepromoted by the incorporation of an arginine-rich peptide, or othermembrane permeable peptide, into the structure of the ligand or RNAbinding protein or attachment of such a peptide to the ligand or RNAbinding protein. See, e.g., U.S. Patent App. Pub. Nos. 20060030003;20060025361; 20060019286; 20060019258. In one aspect, the inventionprovides lipid-based formulations for delivering, e.g., introducingnucleic acids of the invention as nucleic acid-lipid particlescomprising an RNAi molecule to a cell, see .g., U.S. Patent App. Pub.No. 20060008910.

Methods for making and using RNAi molecules, e.g., siRNA and/or miRNA,for selectively degrade RNA are well known in the art, see, e.g., U.S.Pat. No. 6,506,559; 6,511,824; 6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding a xylanase, mannanaseand/or glucanase. These methods can be repeated or used in variouscombinations to generate xylanases and/or glucanases having an alteredor different activity or an altered or different stability from that ofa xylanase, mannanase and/or glucanase encoded by the template nucleicacid. These methods also can be repeated or used in variouscombinations, e.g., to generate variations in gene/message expression,message translation or message stability. In another aspect, the geneticcomposition of a cell is altered by, e.g., modification of a homologousgene ex vivo, followed by its reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly (e.g.,GeneReassembly, see, e.g., U.S. Pat. No. 6,537,776), gene sitesaturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-45′7; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor(1985) “The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor(1985) “The rapid generation of oligonucleotide-directed mutations athigh frequency using phosphorothioate-modified DNA” Nucl. Acids Res. 13:8765-8787 (1985); Nakamaye (1986) “Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis” Nucl. Acids Res.14: 9679-9698; Sayers (1988) “Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis” Nucl. Acids Res. 16:791-802; andSayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer (1988) “Improved enzymatic invitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;”U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application serial no. (USSN) 09/407,800, “SHUFFLING OFCODON ALTERED GENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OFWHOLE CELLS AND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by delCardayre et al., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATEDNUCLEIC ACID RECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714;6,368,861; 6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OFCODON-VARIED OLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welchet al., U.S. Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or acombination thereof are used to modify the nucleic acids of theinvention to generate xylanases and/or glucanases with new or alteredproperties (e.g., activity under highly acidic or alkaline conditions,high or low temperatures, and the like). Polypeptides encoded by themodified nucleic acids can be screened for an activity before testingfor xylan hydrolysis or other activity. Any testing modality or protocolcan be used, e.g., using a capillary array platform. See, e.g., U.S.Pat. Nos. 6,361,974; 6,280,926; 5,939,250.

Gene Site Saturation Mutagenesis, or, GSSM

The invention also provides methods for making enzyme using Gene SiteSaturation mutagenesis, or, GSSM, as described herein, and also in U.S.Pat. Nos. 6,171,820 and 6,579,258. In one aspect, codon primerscontaining a degenerate N,N,G/T sequence are used to introduce pointmutations into a polynucleotide, e.g., a xylanase, mannanase and/orglucanase or an antibody of the invention, so as to generate a set ofprogeny polypeptides in which a full range of single amino acidsubstitutions is represented at each amino acid position, e.g., an aminoacid residue in an enzyme active site or ligand binding site targeted tobe modified. These oligonucleotides can comprise a contiguous firsthomologous sequence, a degenerate N,N,G/T sequence, and, in one aspect,a second homologous sequence. The downstream progeny translationalproducts from the use of such oligonucleotides include all possibleamino acid changes at each amino acid site along the polypeptide,because the degeneracy of the N,N,G/T sequence includes codons for all20 amino acids. In one aspect, one such degenerate oligonucleotide(comprised of, e.g., one degenerate N,N,G/T cassette) is used forsubjecting each original codon in a parental polynucleotide template toa full range of codon substitutions. In another aspect, at least twodegenerate cassettes are used—either in the same oligonucleotide or not,for subjecting at least two original codons in a parental polynucleotidetemplate to a full range of codon substitutions. For example, more thanone N,N,G/T sequence can be contained in one oligonucleotide tointroduce amino acid mutations at more than one site. This plurality ofN,N,G/T sequences can be directly contiguous, or separated by one ormore additional nucleotide sequence(s). In another aspect,oligonucleotides serviceable for introducing additions and deletions canbe used either alone or in combination with the codons containing anN,N,G/T sequence, to introduce any combination or permutation of aminoacid additions, deletions, and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position X 100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can in one aspect be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,xylanases and/or glucanases) molecules such that all 20 natural aminoacids are represented at the one specific amino acid positioncorresponding to the codon position mutagenized in the parentalpolynucleotide (other aspects use less than all 20 naturalcombinations). The 32-fold degenerate progeny polypeptides generatedfrom each saturation mutagenesis reaction vessel can be subjected toclonal amplification (e.g. cloned into a suitable host, e.g., E. colihost, using, e.g., an expression vector) and subjected to expressionscreening. When an individual progeny polypeptide is identified byscreening to display a favorable change in property (when compared tothe parental polypeptide, such as increased xylan hydrolysis activityunder alkaline or acidic conditions), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined −6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,N sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (gene site saturation mutagenesis (GSSM)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,N sequence and preferably but not necessarilya second homologous sequence. The downstream progeny translationalproducts from the use of such oligos include all possible amino acidchanges at each amino acid site along the polypeptide, because thedegeneracy of the N,N,N sequence includes codons for all 20 amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,N cassette) is used for subjecting each original codon in a parentalpolynucleotide template to a full range of codon substitutions. Inanother aspect, at least two degenerate N,N,N cassettes are used—eitherin the same oligo or not, for subjecting at least two original codons ina parental polynucleotide template to a full range of codonsubstitutions. Thus, more than one N,N,N sequence can be contained inone oligo to introduce amino acid mutations at more than one site. Thisplurality of N,N,N sequences can be directly contiguous, or separated byone or more additional nucleotide sequence(s). In another aspect, oligosserviceable for introducing additions and deletions can be used eitheralone or in combination with the codons containing an N,N,N sequence, tointroduce any combination or permutation of amino acid additions,deletions and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,N triplets, i.e. a degenerate (N,N,N)_(n)sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (e.g. in an oligo)a degenerate triplet sequence comprised of only one N, where the N canbe in the first second or third position of the triplet. Any other basesincluding any combinations and permutations thereof can be used in theremaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan in one aspect be used in combination with degenerate primersdisclosed. It is appreciated that in some situations, it is advantageousto use nondegenerate oligos to generate specific point mutations in aworking polynucleotide. This provides a means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

Thus, in one aspect of this invention, each saturation mutagenesisreaction vessel contains polynucleotides encoding at least 20 progenypolypeptide molecules such that all 20 amino acids are represented atthe one specific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined −6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

Thus, in a non-limiting exemplification, this invention provides for theuse of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

In addition to performing mutagenesis along the entire sequence of agene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is preferably everyinteger from 15 to 100,000. Thus, instead of mutagenizing every positionalong a molecule, one can subject every or a discrete number of bases(preferably a subset totaling from 15 to 100,000) to mutagenesis.Preferably, a separate nucleotide is used for mutagenizing each positionor group of positions along a polynucleotide sequence. A group of 3positions to be mutagenized may be a codon. The mutations are preferablyintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Exemplary cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

In a general sense, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette ispreferably about 1-500 bases in length) in defined polynucleotidesequence to be mutagenized (wherein the sequence to be mutagenized ispreferably from about 15 to 100,000 bases in length). Thus, a group ofmutations (ranging from 1 to 100 mutations) is introduced into eachcassette to be mutagenized. A grouping of mutations to be introducedinto one cassette can be different or the same from a second grouping ofmutations to be introduced into a second cassette during the applicationof one round of saturation mutagenesis. Such groupings are exemplifiedby deletions, additions, groupings of particular codons and groupings ofparticular nucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF) and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In one exemplification a grouping of mutations that can be introducedinto a mutagenic cassette, this invention specifically provides fordegenerate codon substitutions (using degenerate oligos) that code for2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20amino acids at each position and a library of polypeptides encodedthereby.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., xylanases and/or glucanases,or antibodies of the invention, with new or altered properties.

SLR is a method of ligating oligonucleotide fragments togethernon-stochastically. This method differs from stochastic oligonucleotideshuffling in that the nucleic acid building blocks are not shuffled,concatenated or chimerized randomly, but rather are assemblednon-stochastically. See, e.g., U.S. Pat. Nos. 6,773,900; 6,740,506;6,713,282; 6,635,449; 6,605,449; 6,537,776. In one aspect, SLRcomprises: (a) providing a template polynucleotide, wherein the templatepolynucleotide comprises sequence encoding a homologous gene; (b)providing a plurality of building block polynucleotides, wherein thebuilding block polynucleotides are designed to cross-over reassemblewith the template polynucleotide at a predetermined sequence, and abuilding block polynucleotide comprises a sequence that is a variant ofthe homologous gene and a sequence homologous to the templatepolynucleotide flanking the variant sequence; (c) combining a buildingblock polynucleotide with a template polynucleotide such that thebuilding block polynucleotide cross-over reassembles with the templatepolynucleotide to generate polynucleotides comprising homologous genesequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are preferably shared by at leasttwo of the progenitor templates. The demarcation points can thereby beused to delineate the boundaries of oligonucleotide building blocks tobe generated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

Synthetic Gene Reassembly

In one aspect, the present invention provides a non-stochastic methodtermed synthetic gene reassembly (e.g., GeneReassembly, see, e.g., U.S.Pat. No. 6,537,776), which differs from stochastic shuffling in that thenucleic acid building blocks are not shuffled or concatenated orchimerized randomly, but rather are assembled non-stochastically.

The synthetic gene reassembly method does not depend on the presence ofa high level of homology between polynucleotides to be shuffled. Theinvention can be used to non-stochastically generate libraries (or sets)of progeny molecules comprised of over 10¹⁰⁰ different chimeras.Conceivably, synthetic gene reassembly can even be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras.

Thus, in one aspect, the invention provides a non-stochastic method ofproducing a set of finalized chimeric nucleic acid molecules having anoverall assembly order that is chosen by design, which method iscomprised of the steps of generating by design a plurality of specificnucleic acid building blocks having serviceable mutually compatibleligatable ends and assembling these nucleic acid building blocks, suchthat a designed overall assembly order is achieved.

In one aspect, synthetic gene reassembly comprises a method of: 1)preparing a progeny generation of molecule(s) (including a moleculecomprising a polynucleotide sequence, e.g., a molecule comprising apolypeptide coding sequence), that is mutagenized to achieve at leastone point mutation, addition, deletion, &/or chimerization, from one ormore ancestral or parental generation template(s); 2) screening theprogeny generation molecule(s), e.g., using a high throughput method,for at least one property of interest (such as an improvement in anenzyme activity); 3) in one aspect obtaining &/or cataloguing structural&/or and functional information regarding the parental &/or progenygeneration molecules; and 4) in one aspect repeating any of steps 1) to3). In one aspect, there is generated (e.g., from a parentpolynucleotide template), in what is termed “codon site-saturationmutagenesis,” a progeny generation of polynucleotides, each having atleast one set of up to three contiguous point mutations (i.e. differentbases comprising a new codon), such that every codon (or every family ofdegenerate codons encoding the same amino acid) is represented at eachcodon position. Corresponding to, and encoded by, this progenygeneration of polynucleotides, there is also generated a set of progenypolypeptides, each having at least one single amino acid point mutation.In a one aspect, there is generated, in what is termed “amino acidsite-saturation mutagenesis”, one such mutant polypeptide for each ofthe 19 naturally encoded polypeptide-forming alpha-amino acidsubstitutions at each and every amino acid position along thepolypeptide. This yields, for each and every amino acid position alongthe parental polypeptide, a total of 20 distinct progeny polypeptidesincluding the original amino acid, or potentially more than 21 distinctprogeny polypeptides if additional amino acids are used either insteadof or in addition to the 20 naturally encoded amino acids

Thus, in another aspect, this approach is also serviceable forgenerating mutants containing, in addition to &/or in combination withthe 20 naturally encoded polypeptide-forming alpha-amino acids, otherrare &/or not naturally-encoded amino acids and amino acid derivatives.In yet another aspect, this approach is also serviceable for generatingmutants by the use of, in addition to &/or in combination with naturalor unaltered codon recognition systems of suitable hosts, altered,mutagenized, &/or designer codon recognition systems (such as in a hostcell with one or more altered tRNA molecules.

In yet another aspect, this invention relates to recombination and morespecifically to a method for preparing polynucleotides encoding apolypeptide by a method of in vivo re-assortment of polynucleotidesequences containing regions of partial homology, assembling thepolynucleotides to form at least one polynucleotide and screening thepolynucleotides for the production of polypeptide(s) having a usefulproperty.

In yet another aspect, this invention is serviceable for analyzing andcataloguing, with respect to any molecular property (e.g. an enzymaticactivity) or combination of properties allowed by current technology,the effects of any mutational change achieved (including particularlysaturation mutagenesis). Thus, a comprehensive method is provided fordetermining the effect of changing each amino acid in a parentalpolypeptide into each of at least 19 possible substitutions. This allowseach amino acid in a parental polypeptide to be characterized andcatalogued according to its spectrum of potential effects on ameasurable property of the polypeptide.

In one aspect, an intron may be introduced into a chimeric progenymolecule by way of a nucleic acid building block. Introns often haveconsensus sequences at both termini in order to render them operational.In addition to enabling gene splicing, introns may serve an additionalpurpose by providing sites of homology to other nucleic acids to enablehomologous recombination. For this purpose, and potentially others, itmay be sometimes desirable to generate a large nucleic acid buildingblock for introducing an intron. If the size is overly large easilygenerating by direct chemical synthesis of two single stranded oligos,such a specialized nucleic acid building block may also be generated bydirect chemical synthesis of more than two single stranded oligos or byusing a polymerase-based amplification reaction

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in one aspect, the overall assembly order inwhich the nucleic acid building blocks can be coupled is specified bythe design of the ligatable ends and, if more than one assembly step isto be used, then the overall assembly order in which the nucleic acidbuilding blocks can be coupled is also specified by the sequential orderof the assembly step(s). In a one aspect of the invention, the annealedbuilding pieces are treated with an enzyme, such as a ligase (e.g., T4DNA ligase) to achieve covalent bonding of the building pieces.

Coupling can occur in a manner that does not make use of everynucleotide in a participating overhang. The coupling is particularlylively to survive (e.g. in a transformed host) if the couplingreinforced by treatment with a ligase enzyme to form what may bereferred to as a “gap ligation” or a “gapped ligation”. This type ofcoupling can contribute to generation of unwanted background product(s),but it can also be used advantageously increase the diversity of theprogeny library generated by the designed ligation reassembly. Certainoverhangs are able to undergo self-coupling to form a palindromiccoupling. A coupling is strengthened substantially if it is reinforcedby treatment with a ligase enzyme. Lack of 5′ phosphates on theseoverhangs can be used advantageously to prevent this type of palindromicself-ligation. Accordingly, this invention provides that nucleic acidbuilding blocks can be chemically made (or ordered) that lack a 5′phosphate group. Alternatively, they can be removed, e.g. by treatmentwith a phosphatase enzyme, such as a calf intestinal alkalinephosphatase (CIAP), in order to prevent palindromic self-ligations inligation reassembly processes.

In a another aspect, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes. Thexylanases and/or glucanases of the present invention can be mutagenizedin accordance with the methods described herein.

Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates (e.g., polynucleotides ofThe invention) are aligned in order to select one or more demarcationpoints, which demarcation points can be located at an area of homology.The demarcation points can be used to delineate the boundaries ofnucleic acid building blocks to be generated. Thus, the demarcationpoints identified and selected in the progenitor molecules serve aspotential chimerization points in the assembly of the progeny molecules.

Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates and preferably at almost all of theprogenitor templates. Even more preferably still a serviceabledemarcation point is an area of homology that is shared by all of theprogenitor templates.

In a one aspect, the gene reassembly process is performed exhaustivelyin order to generate an exhaustive library. In other words, all possibleordered combinations of the nucleic acid building blocks are representedin the set of finalized chimeric nucleic acid molecules. At the sametime, the assembly order (i.e. the order of assembly of each buildingblock in the 5′ to 3 sequence of each finalized chimeric nucleic acid)in each combination is by design (or non-stochastic). Because of thenon-stochastic nature of the method, the possibility of unwanted sideproducts is greatly reduced.

In another aspect, the method provides that the gene reassembly processis performed systematically, for example to generate a systematicallycompartmentalized library, with compartments that can be screenedsystematically, e.g., one by one. In other words the invention providesthat, through the selective and judicious use of specific nucleic acidbuilding blocks, coupled with the selective and judicious use ofsequentially stepped assembly reactions, an experimental design can beachieved where specific sets of progeny products are made in each ofseveral reaction vessels. This allows a systematic examination andscreening procedure to be performed. Thus, it allows a potentially verylarge number of progeny molecules to be examined systematically insmaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant gene reassembly invention, theprogeny molecules generated preferably comprise a library of finalizedchimeric nucleic acid molecules having an overall assembly order that ischosen by design. In a particularly aspect, such a generated library iscomprised of greater than 10³ to greater than 10¹⁰⁰⁰ different progenymolecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. According to one aspect, this polynucleotide is a gene,which may be a man-made gene. According to another aspect, thispolynucleotide is a gene pathway, which may be a man-made gene pathway.The invention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of the step in whichthe building blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be in oneaspect removed in an in vitro process (e.g., by mutagenesis) or in an invivo process (e.g., by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, according to another aspect, the invention provides that a nucleicacid building block can be used to introduce an intron. Thus, theinvention provides that functional introns may be introduced into aman-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). Preferably, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In a one aspect,the recombination is facilitated by, or occurs at, areas of homologybetween the man-made, intron-containing gene and a nucleic acid, whichserves as a recombination partner. In one aspect, the recombinationpartner may also be a nucleic acid generated by the invention, includinga man-made gene or a man-made gene pathway. Recombination may befacilitated by or may occur at areas of homology that exist at the one(or more) artificially introduced intron(s) in the man-made gene.

The synthetic gene reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which preferably hastwo ligatable ends. The two ligatable ends on each nucleic acid buildingblock may be two blunt ends (i.e. each having an overhang of zeronucleotides), or preferably one blunt end and one overhang, or morepreferably still two overhangs.

A useful overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

In one aspect, a nucleic acid building block is generated by chemicalsynthesis of two single-stranded nucleic acids (also referred to assingle-stranded oligos) and contacting them so as to allow them toanneal to form a double-stranded nucleic acid building block.

A double-stranded nucleic acid building block can be of variable size.The sizes of these building blocks can be small or large. Exemplarysizes for building block range from 1 base pair (not including anyoverhangs) to 100,000 base pairs (not including any overhangs). Otherexemplary size ranges are also provided, which have lower limits of from1 bp to 10,000 bp (including every integer value in between) and upperlimits of from 2 bp to 100,000 bp (including every integer value inbetween).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan.

According to one aspect, a double-stranded nucleic acid building blockis generated by first generating two single stranded nucleic acids andallowing them to anneal to form a double-stranded nucleic acid buildingblock. The two strands of a double-stranded nucleic acid building blockmay be complementary at every nucleotide apart from any that form anoverhang; thus containing no mismatches, apart from any overhang(s).According to another aspect, the two strands of a double-strandednucleic acid building block are complementary at fewer than everynucleotide apart from any that form an overhang. Thus, according to thisaspect, a double-stranded nucleic acid building block can be used tointroduce codon degeneracy. The codon degeneracy can be introduced usingthe site-saturation mutagenesis described herein, using one or moreN,N,G/T cassettes or alternatively using one or more N,N,N cassettes.

The in vivo recombination method of the invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

In one aspect the invention described herein is directed to the use ofrepeated cycles of reductive reassortment, recombination and selectionwhich allow for the directed molecular evolution of highly complexlinear sequences, such as DNA, RNA or proteins thorough recombination.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,xylanases and/or glucanases, or antibodies of the invention, with new oraltered properties. Optimized directed evolution is directed to the useof repeated cycles of reductive reassortment, recombination andselection that allow for the directed molecular evolution of nucleicacids through recombination. Optimized directed evolution allowsgeneration of a large population of evolved chimeric sequences, whereinthe generated population is significantly enriched for sequences thathave a predetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a 1/3 chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB™ (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new xylanase, mannanase and/or glucanase phenotype isidentified, re-isolated, again modified, re-tested for activity. Thisprocess can be iteratively repeated until a desired phenotype isengineered. For example, an entire biochemical anabolic or catabolicpathway can be engineered into a cell, including, e.g., xylanase,mannanase and/or glucanase activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new xylanase, mannanaseand/or glucanase phenotype), it can be removed as a variable bysynthesizing larger parental oligonucleotides that include the sequenceto be removed. Since incorporating the sequence within a larger sequenceprevents any crossover events, there will no longer be any variation ofthis sequence in the progeny polynucleotides. This iterative practice ofdetermining which oligonucleotides are most related to the desiredtrait, and which are unrelated, allows more efficient exploration all ofthe possible protein variants that might be provide a particular traitor activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,xylanases, and the like. In vivo shuffling can be performed utilizingthe natural property of cells to recombine multimers. Whilerecombination in vivo has provided the major natural route to moleculardiversity, genetic recombination remains a relatively complex processthat involves 1) the recognition of homologies; 2) strand cleavage,strand invasion, and metabolic steps leading to the production ofrecombinant chiasma; and finally 3) the resolution of chiasma intodiscrete recombined molecules. The formation of the chiasma requires therecognition of homologous sequences.

In another aspect, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The invention canrely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process.

Therefore, in another aspect of the invention, novel polynucleotides canbe generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

a) Primers that include a poly-A head and poly-T tail which when madesingle-stranded would provide orientation can be utilized. This isaccomplished by having the first few bases of the primers made from RNAand hence easily removed RNAseH.

b) Primers that include unique restriction cleavage sites can beutilized. Multiple sites, a battery of unique sequences and repeatedsynthesis and ligation steps would be required.

c) The inner few bases of the primer could be thiolated and anexonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (RI). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by:

1) The use of vectors only stably maintained when the construct isreduced in complexity.

2) The physical recovery of shortened vectors by physical procedures. Inthis case, the cloning vector would be recovered using standard plasmidisolation procedures and size fractionated on either an agarose gel, orcolumn with a low molecular weight cut off utilizing standardprocedures.

3) The recovery of vectors containing interrupted genes which can beselected when insert size decreases.

4) The use of direct selection techniques with an expression vector andthe appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates a method of the invention. Encodingnucleic acid sequences (quasi-repeats) derived from three (3) uniquespecies are described. Each sequence encodes a protein with a distinctset of properties. Each of the sequences differs by a single or a fewbase pairs at a unique position in the sequence. The quasi-repeatedsequences are separately or collectively amplified and ligated intorandom assemblies such that all possible permutations and combinationsare available in the population of ligated molecules. The number ofquasi-repeat units can be controlled by the assembly conditions. Theaverage number of quasi-repeated units in a construct is defined as therepetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector and transfected into an appropriate host cell. The cells are thenpropagated and “reductive reassortment” is effected. The rate of thereductive reassortment process may be stimulated by the introduction ofDNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

In one aspect (optionally), the method comprises the additional step ofscreening the library members of the shuffled pool to identifyindividual shuffled library members having the ability to bind orotherwise interact, or catalyze a particular reaction (e.g., such ascatalytic domain of an enzyme) with a predetermined macromolecule, suchas for example a proteinaceous receptor, an oligosaccharide, virion, orother predetermined compound or structure.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes (e.g., catalysts,solutes for increasing osmolarity of an aqueous solution and the like)and/or can be subjected to one or more additional cycles of shufflingand/or selection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)—CC-1065, or a synthetic analog such as (+)—CC-1065-(N-3-Adenine (SeeSun and Hurley, (1992); an N-acetylated or deacetylated4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See™, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[α]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[α]pyrene-7,8-dihydrodio1-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”) andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Exemplary means for slowing or halting PCRamplification consist of UV light (+)—CC-1065 and(+)—CC-1065-(N-3-Adenine). Particularly encompassed means are DNAadducts or polynucleotides comprising the DNA adducts from thepolynucleotides or polynucleotides pool, which can be released orremoved by a process including heating the solution comprising thepolynucleotides prior to further processing.

In another aspect the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (e.g., xylanase) sequences of theinvention. The invention also provides additional methods for isolatingxylanases using the nucleic acids and polypeptides of the invention. Inone aspect, the invention provides for variants of a xylanase codingsequence (e.g., a gene, cDNA or message) of the invention, which can bealtered by any means, including, e.g., random or stochastic methods, or,non-stochastic, or “directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generate newnucleic acids which encode polypeptides having characteristics whichenhance their value in industrial, medical, laboratory (research),pharmaceutical, food and feed and food and feed supplement processingand other applications and processes. In such procedures, a large numberof variant sequences having one or more nucleotide differences withrespect to the sequence obtained from the natural isolate are generatedand characterized. These nucleotide differences can result in amino acidchanges with respect to the polypeptides encoded by the nucleic acidsfrom the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 (moles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids areevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in PCTPublication No. WO 91/16427, published Oct. 31, 1991, entitled “Methodsfor Phenotype Creation from Multiple Gene Populations”.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in Arkin, A. P. and Youvan, D.C., PNAS, USA,89:7811-7815, 1992.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described inDelegrave, S, and Youvan, D.C., Biotechnology Research, 11:1548-1552,1993. Random and site-directed mutagenesis are described in Arnold, F.H., Current Opinion in Biotechnology, 4:450-455, 1993.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in U.S. Pat.No. 5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassemblyby Interrupting Synthesis” and U.S. Pat. No. 5,939,250, filed May 22,1996, entitled, “Production of Enzymes Having Desired Activities byMutagenesis.

The variants of the polypeptides of the invention may be variants inwhich one or more of the amino acid residues of the polypeptides of theinvention are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Alanine,Valine, Leucine and Isoleucine with another aliphatic amino acid;replacement of a Serine with a Threonine or vice versa; replacement ofan acidic residue such as Aspartic acid and Glutamic acid with anotheracidic residue; replacement of a residue bearing an amide group, such asAsparagine and Glutamine, with another residue bearing an amide group;exchange of a basic residue such as Lysine and Arginine with anotherbasic residue; and replacement of an aromatic residue such asPhenylalanine, Tyrosine with another aromatic residue.

Other variants are those in which one or more of the amino acid residuesof the polypeptides of the invention includes a substituent group.

Still other variants are those in which the polypeptide is associatedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol).

Additional variants are those in which additional amino acids are fusedto the polypeptide, such as a leader sequence, a secretory sequence, aproprotein sequence or a sequence which facilitates purification,enrichment, or stabilization of the polypeptide. In some aspects, thefragments, derivatives and analogs retain the same biological functionor activity as the polypeptides of the invention and sequencessubstantially identical thereto. In other aspects, the fragment,derivative, or analog includes a proprotein, such that the fragment,derivative, or analog can be activated by cleavage of the proproteinportion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying xylanase-encoding nucleicacids to modify codon usage. In one aspect, the invention providesmethods for modifying codons in a nucleic acid encoding a xylanase toincrease or decrease its expression in a host cell. The invention alsoprovides nucleic acids encoding a xylanase modified to increase itsexpression in a host cell, xylanase so modified, and methods of makingthe modified xylanases. The method comprises identifying a“non-preferred” or a “less preferred” codon in xylanase-encoding nucleicacid and replacing one or more of these non-preferred or less preferredcodons with a “preferred codon” encoding the same amino acid as thereplaced codon and at least one non-preferred or less preferred codon inthe nucleic acid has been replaced by a preferred codon encoding thesame amino acid. A preferred codon is a codon over-represented in codingsequences in genes in the host cell and a non-preferred or lesspreferred codon is a codon under-represented in coding sequences ingenes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli and Pseudomonas fluorescens; gram positive bacteria, such asLactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris,Bacillus subtilis. Exemplary host cells also include eukaryoticorganisms, e.g., various yeast, such as Saccharomyces sp., includingSaccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris,and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, andmammalian cells and cell lines and insect cells and cell lines. Otherexemplary host cells include bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, fungal cells, such as Aspergillus, yeast such as anyspecies of Pichia, Saccharomyces, Schizosaccharomyces, Schwanniomyces,including Pichia pastoris, Saccharomyces cerevisiae, orSchizosaccharomyces pombe, insect cells such as Drosophila S2 andSpodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art. Thus, the invention also includesnucleic acids and polypeptides optimized for expression in theseorganisms and species.

For example, the codons of a nucleic acid encoding a xylanase isolatedfrom a bacterial cell are modified such that the nucleic acid isoptimally expressed in a bacterial cell different from the bacteria fromwhich the xylanase was derived, a yeast, a fungi, a plant cell, aninsect cell or a mammalian cell. Methods for optimizing codons are wellknown in the art, see, e.g., U.S. Pat. No. 5,795,737; Baca (2000) Int.J. Parasitol. 30:113-118; Hale (1998) Protein Expr. Purif. 12:185-188;Narum (2001) Infect. Immun. 69:7250-7253. See also Narum (2001) Infect.Immun. 69:7250-7253, describing optimizing codons in mouse systems;Outchkourov (2002) Protein Expr. Purif. 24:18-24, describing optimizingcodons in yeast; Feng (2000) Biochemistry 39:15399-15409, describingoptimizing codons in E. coli; Humphreys (2000) Protein Expr. Purif.20:252-264, describing optimizing codon usage that affects secretion inE. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (e.g., a xylanase), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides methods of making and using these transgenic non-humananimals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats, horses, dogs, fish and mice, comprising the nucleicacids of the invention. These animals can be used, e.g., as in vivomodels to study xylanase activity, or, as models to screen for agentsthat change the xylanase activity in vivo. The coding sequences for thepolypeptides to be expressed in the transgenic non-human animals can bedesigned to be constitutive, or, under the control of tissue-specific,developmental-specific or inducible transcriptional regulatory factors.Transgenic non-human animals can be designed and generated using anymethod known in the art; see, e.g., U.S. Pat. Nos. 6,211,428; 6,187,992;6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933; 5,387,742;5,087,571, describing making and using transformed cells and eggs andtransgenic mice, rats, rabbits, sheep, pigs, chickens, goats, fish andcows. See also, e.g., Pollock (1999) J. Immunol. Methods 231:147-157,describing the production of recombinant proteins in the milk oftransgenic dairy animals; Baguisi (1999) Nat. Biotechnol. 17:456-461,demonstrating the production of transgenic goats. U.S. Pat. No.6,211,428, describes making and using transgenic non-human mammals whichexpress in their brains a nucleic acid construct comprising a DNAsequence. U.S. Pat. No. 5,387,742, describes injecting clonedrecombinant or synthetic DNA sequences into fertilized mouse eggs,implanting the injected eggs in pseudo-pregnant females, and growing toterm transgenic mice whose cells express proteins related to thepathology of Alzheimer's disease. U.S. Pat. No. 6,187,992, describesmaking and using a transgenic mouse whose genome comprises a disruptionof the gene encoding amyloid precursor protein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing a xylanase of the invention, or, a fusion proteincomprising a xylanase of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., a xylanase), an expression cassette or vectoror a transfected or transformed cell of the invention. The inventionalso provides plant products or byproducts, e.g., fruits, oils, seeds,leaves, extracts and the like, including any plant part, comprising anucleic acid and/or a polypeptide (e.g., a xylanase) of the invention,e.g., wherein the nucleic acid or polypeptide of the invention isheterologous to the plant, plant part, seed etc. The transgenic plant(which includes plant parts, fruits, seeds etc.) can be dicotyledonous(a dicot) or monocotyledonous (a monocot). The invention also providesmethods of making and using these transgenic plants and seeds. Thetransgenic plant or plant cell expressing a polypeptide of the presentinvention may be constructed in accordance with any method known in theart. See, for example, U.S. Pat. No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's xylanase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on starch-producing plants, such aspotato, wheat, rice, barley, and the like. Nucleic acids of theinvention can be used to manipulate metabolic pathways of a plant inorder to optimize or alter host's expression of xylanase. The can changexylanase activity in a plant. Alternatively, a xylanase of the inventioncan be used in production of a transgenic plant to produce a compoundnot naturally produced by that plant. This can lower production costs orcreate a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, in one aspect (optionally), marker genesinto a target expression construct (e.g., a plasmid), along withpositioning of the promoter and the terminator sequences. This caninvolve transferring the modified gene into the plant through a suitablemethod. For example, a construct may be introduced directly into thegenomic DNA of the plant cell using techniques such as electroporationand microinjection of plant cell protoplasts, or the constructs can beintroduced directly to plant tissue using ballistic methods, such as DNAparticle bombardment. For example, see, e.g., Christou (1997) Plant Mol.Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein(1987) Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69,discussing use of particle bombardment to introduce transgenes intowheat; and Adam (1997) supra, for use of particle bombardment tointroduce YACs into plant cells. For example, Rinehart (1997) supra,used particle bombardment to generate transgenic cotton plants.Apparatus for accelerating particles is described U.S. Pat. No.5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000particle acceleration instrument; see also, John, U.S. Pat. No.5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describingparticle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci. USA80:4803; Thykjaer (1997) supra; Park (1996) Plant Mol. Biol.32:1135-1148, discussing T-DNA integration into genomic DNA. See alsoD'Halluin, U.S. Pat. No. 5,712,135, describing a process for the stableintegration of a DNA comprising a gene that is functional in a cell of acereal, or other monocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., axylanase) of the invention. The desired effects can be passed to futureplant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.Transgenic plants and seeds of the invention can be any monocot ordicot, e.g., a monocot corn, sugarcane, rice, wheat, barley, switchgrassor Miscanthus; or a dicot oilseed crop, soy, canola, rapeseed, flax,cotton, palm oil, sugar beet, peanut, tree, poplar or lupine.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants (and/or their seeds) which contain fiber cells,including, e.g., cotton, silk cotton tree (Kapok, Ceiba pentandra),desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp,roselle, jute, sisal abaca and flax. In alternative embodiments, thetransgenic plants of the invention can be members of the genusGossypium, including members of any Gossypium species, such as G.arboreum;. G. herbaceum, G. barbadense, and G. hirsutum.

The invention also provides for transgenic plants (and/or their seeds)to be used for producing large amounts of the polypeptides (e.g., axylanase or antibody) of the invention. For example, see Palmgren (1997)Trends Genet. 13:348; Chong (1997) Transgenic Res. 6:289-296 (producinghuman milk protein beta-casein in transgenic potato plants using anauxin-inducible, bidirectional mannopine synthase (mas 1′,2′) promoterwith Agrobacterium tumefaciens-mediated leaf disc transformationmethods).

Using known procedures, one of skill can screen for plants (and/or theirseeds) of the invention by detecting the increase or decrease oftransgene mRNA or protein in transgenic plants. Means for detecting andquantitation of mRNAs or proteins are well known in the art.

Polypeptides and Peptides

In one aspect, the invention provides isolated, synthetic or recombinantpolypeptides and peptides having xylanase, a mannanase and/or aglucanase activity, or polypeptides and peptides capable of generatingan antibody that specifically binds to a xylanase or a glucanase,including an enzyme of this invention, including the amino acidsequences of the invention, which include those having at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100% (complete) sequenceidentity to an exemplary polypeptide of the invention (as defined above,including SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22 and SEQ ID NO:24), or any polypeptide of thisinvention, including for example SEQ ID NO:2 having one or more aminoacid residue changes (mutations) as set forth in Table 1 and asdescribed herein, also including a genus of polypeptides having varioussequence identities based on the exemplary SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24; andthese exemplary polypeptides have the following enzymatic activity(e.g., the xylanase of SEQ ID NO:2, is encoded e.g., by SEQ ID NO:1; thearabinofuranosidase of SEQ ID NO:14, is encoded e.g., by SEQ ID NO:13,and the like):

SEQ ID NO: Name Activity 1, 2 xyl 11 Xylanase 11, 12 control xylanaseXylanase 13, 14 Arabinofuranosidase 15, 16 Xylanase 17, 18 Oligomerase19, 20 B-glucosidase 21, 22 Arabinofuranosidase 23, 24 Beta-xylosidase3, 4 Endoglucanase 5, 6 Oligomerase 7, 8 Cellobiohydrolase  9, 10Cellobiohydrolase

The invention also provides enzyme-encoding nucleic acids with a commonnovelty in that they encode a subset of xylanases, or a Glade,comprising the “X14 module”. In one aspect, the invention also providesenzyme-encoding nucleic acids with a common novelty in that they encodea Glade comprising the “X14 module” (see, e.g., J. Bacteriol. 2002August; 184(15): 4124-4133). X14-comprising xylanase members include SEQID NO:2 having one or more amino acid residue changes (mutations) as setforth in Table 1 and as described herein.

In one aspect, the invention provides chimeric enzymes, includingxylanases, glucanases and/or glycosidases, having heterologouscarbohydrate-binding modules (CBMs), e.g., for use in the processes ofthe invention and in various industrial, medical, pharmaceutical,research, food and feed and food and feed supplement processing andother applications. For example, in one aspect the invention providesenzymes, e.g., hydrolases, including glycosyl hydrolases (such asxylanases, glucanases) comprising one or more CBMs of an enzyme of theinvention, including the CBM-like X14 module discussed above. In anotheraspect, CBMs, e.g., X14 modules, between different enzymes of theinvention can be swapped; or, alternatively, one or more CBMs of one ormore enzymes of the invention can be spliced into an enzyme, e.g., ahydrolase, e.g., any glycosyl hydrolase, such as a xylanase.

Glycosyl hydrolases that utilize insoluble substrates are modular,usually comprising catalytic modules appended to one or morenon-catalytic carbohydrate-binding modules (CBMs). In nature, CBMs arethought to promote the interaction of the glycosyl hydrolase with itstarget substrate polysaccharide. For example, as discussed above, X14 isa xylan binding module. Thus, the invention provides chimeric enzymeshaving heterologous, non-natural substrates; including chimeric enzymeshaving multiple substrates by nature of their “spliced-in” heterologousCBMs, e.g., a spliced-in X14 module of the invention—thus giving thechimeric enzyme new specificity for xylan and galactan, or enhancedbinding to xylan and galactan. The heterologous CBMs of the chimericenzymes of the invention can be designed to be modular, i.e., to beappended to a catalytic module or catalytic domain (e.g., an activesite), which also can be heterologous or can be homologous to theenzyme.

Utilization of just the catalytic module of a xylanase or a glucanase(e.g., an enzyme of the invention) has been shown to be effective. Thus,the invention provides peptides and polypeptides consisting of, orcomprising, modular CBM/active site modules (e.g., X14), which can behomologously paired or joined as chimeric (heterologous) active site-CBMpairs. Thus, these chimeric polypeptides/peptides of the invention canbe used to improve or alter the performance of an individual enzyme,e.g., a xylanase enzyme. A chimeric catalytic module of the invention(comprising, e.g., at least one CBM of the invention, e.g., X14) can bedesigned to target the enzyme to particular regions of a substrate,e.g., to particular regions of a pulp. For example, in one aspect, thisis achieved by making fusions of the xylanase and various CBMs (either axylanase of the invention with a heterologous CBM, or, a CBM of theinvention with another enzyme, e.g., a hydrolase, such as a xylanase.For example, CBM4, CBM6, and CBM22 are known to bind xylan and mayenhance the effectiveness of the xylanase in pulp biobleaching (see,e.g., Czjzek (2001) J. Biol. Chem. 276(51):48580-7, noting that CBM4,CBM6, and CBM22 are related and CBM interact primarily with xylan). Inanother embodiment, fusion of xylanase and CBM3a or CBM3b, which bindcrystalline cellulose, may help the xylanase penetrate the complexpolysaccharide matrix of pulp and reach inaccessible xylans. Any CBM canbe used to practice the instant invention, e.g., as reviewed by Boraston(2004) Biochem. J. 382:769-781:

Family Protein PDB code CBM1 Cellulase 7A 1CBH (Trichoderma reesei) CBM2Xylanase 10A 1EXG (Cellulomonas fimi) Xylanase 11A 2XBD (Cellulomonasfimi) Xylanase 11A 1HEH (Cellulomonas fimi) CBM3 Scaffoldin 1G43(Clostridium cellulolyticum) Scaffoldin 1NBC (Clostridium thermocellum)Cellulase 9A 1TF4 (Thermobifida fusca) CBM4 Laminarinase 16A 1GUI(Thermotoga maritima) Cellulase 9B 1ULO; (Cellulomonas fimi) 1GU3Cellulase 9B 1CX1 (Cellulomonas fimi) Xylanase 10A 1K45 (Rhodothermusmarinus) CBM5 Cellulase 5A 1AIW (Erwinia chrysanthemi) Chitinase B 1E15(Serratia marcescens) CBM6 Xylanase 11A 1UXX (Clostridium thermocellum)Xylanase 11A 1NAE (Clostridium stercorarium) Xylanase 11A 1UY4(Clostridium stercorarium) Endoglucanase 5A 1UZ0 (Cellvibrio mixtus)CBM9 Xylanase 10A 1I8A (Thermotoga maritima) CBM10 Xylanase 10A 1QLD(Cellvibrio japonicus) CBM12 Chitinase Chi1 1ED7 (Bacillus circulans)CBM13* Xylanase 10A 1XYF (Streptomyces olivaceoviridis) Xylanase 10A1MC9 (Streptomyces lividans) Ricin toxin B-chain 2AAI (Ricinus communis)Abrin 1ABR (Abrus precatorius) CBM14 Tachycitin 1DQC (Tachypleustridentatus) CBM15 Xylanase 10C 1GNY (Cellvibrio japonicus) CBM17Cellulase 5A 1J83 (Clostridium cellulovorans) CBM18* Agglutinin 1WGC(Triticum aestivum) Antimicrobial peptide 1MMC (Amaranthus caudatus)Chitinase/agglutinin 1EIS (Urtica dioica) CBM20* Glucoamylase 1AC0(Aspergillus niger) β-amylase 1CQY (Bacillus cereus) CBM22 Xylanase 10B1DYO (Clostridium thermocellum) CBM27 Mannanase 5A 1OF4 (Thermotogamaritima) CBM28 Cellulase 5A 1UWW (Bacillus sp. 1139) CBM29Non-catalytic protein 1 1GWK (Pyromyces equi) CBM32 Sialidase 33A 1EUU(Micromonospora viridifaciens) Galactose oxidase 1GOF (Cladobotryumdendroides) CBM34* α-Amylase 13A 1UH2 (Thermoactinomyces vulgaris)Neopullulanase 1J0H (Geobacillus stearothermophilus) CBM36 Xylanase 43A1UX7 (Paenibacillus polymyxa) *These families contain too many structureentries to list them all so only representatives are given.

Thus, the invention provides chimeric hydrolases, e.g., a fusion of aglycosidase with different (e.g., heterologous) CBMs to target theenzyme to particular insoluble polysaccharides to enhance performance inan application. In one aspect, the chimeric glycosidase comprises anenzyme of the invention. In one aspect, the chimeric enzyme comprisesfusions of different CBMs to enhance pulp biobleaching performance,e.g., to achieve greater percentage reduction of bleaching chemicals.The invention also provides methods comprising recombining differentCBMs with different xylanases (e.g., CBMs of the invention and/orxylanases of the invention) and screening the resultant chimerics tofind the best combination for a particular application or substrate.

Other variations also are within the scope of this invention, e.g.,where one, two, three, four or five or more residues are removed fromthe carboxy- or amino-terminal ends of any polypeptide of the invention.Another variation includes modifying any residue to increase or decreasepI of a polypeptide, e.g., removing or modifying (e.g., to another aminoacid) a glutamate. This method was used as a general scheme forimproving the enzyme's properties without creating regulatory issuessince no amino acids are mutated; and this general scheme can be usedwith any polypeptide of the invention.

The invention provides isolated, synthetic or recombinant polypeptideshaving xylanase activity, wherein the polypeptide has a sequencemodification of any polypeptide of the invention, including anyexemplary amino acid sequence of the invention, including SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, and also including SEQ ID NO:2 having amino acid residue changes(mutations) as set forth in Table 1 and as described herein. Thesequence change(s) can also comprise any amino acid modification tochange the pI of a polypeptide, e.g., deletion or modification of aglutamate, or changing from a glutamate to another residue.

The invention further provides isolated, synthetic or recombinantpolypeptides having a sequence identity (e.g., at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity) to an exemplary sequence of the invention.

In one aspect, the polypeptide has a xylanase or a glucanase activity;for example, wherein the xylanase activity can comprise hydrolyzing aglycosidic bond in a polysaccharide, e.g., a xylan. In one aspect, thepolypeptide has a xylanase activity comprising catalyzing hydrolysis ofinternal β-1,4-xylosidic linkages. In one aspect, the xylanase activitycomprises an endo-1,4-beta-xylanase activity. In one aspect, thexylanase activity comprises hydrolyzing a xylan to produce a smallermolecular weight xylose and xylo-oligomer. In one aspect, the xylancomprises an arabinoxylan, such as a water soluble arabinoxylan.

The invention provides polypeptides having glucanase activity. In oneaspect, the glucanase activity of a polypeptide or peptide of theinvention (which includes a protein or peptide encoded by a nucleic acidof the invention) comprises an endoglucanase activity, e.g., endo-1,4-and/or 1,3-beta-D-glucan 4-glucano hydrolase activity. In one aspect,the endoglucanase activity comprises catalyzing hydrolysis of1,4-beta-D-glycosidic linkages. In one aspect, the glucanase, e.g.,endoglucanase, activity comprises an endo-1,4- and/or1,3-beta-endoglucanase activity or endo-β-1,4-glucanase activity. In oneaspect, the glucanase activity (e.g., endo-1,4-beta-D-glucan 4-glucanohydrolase activity) comprises hydrolysis of 1,4-beta-D-glycosidiclinkages in cellulose, cellulose derivatives (e.g., carboxy methylcellulose and hydroxy ethyl cellulose) lichenin, beta-1,4 bonds in mixedbeta-1,3 glucans, such as cereal beta-D-glucans and other plant materialcontaining cellulosic parts. In one aspect, the glucanase, xylanase, ormannanase activity comprises hydrolyzing a glucan or otherpolysaccharide to produce a smaller molecular weight polysaccharide oroligomer. In one aspect, the glucan comprises a beta-glucan, such as awater soluble beta-glucan.

The invention provides polypeptides having mannanase (e.g.,endo-1,4-beta-D-mannanase) activity, for example, catalyzing thehydrolysis of a beta-1,4-mannan, e.g., an unsubstituted linearbeta-1,4-mannan. Mannanase activity determination can be determinedusing any known methods, e.g., the Congo Red method, as described e.g.,by Downie (1994) “A new assay for quantifying endo-beta-mannanaseactivity using Congo red dye. Phytochemistry, July 1994, vol. 36, no. 4,p. 829-835; or, as described in U.S. Pat. No. 6,060,299, e.g., byapplying a solution to be tested to 4 mm diameter holes punched out inagar plates containing 0.2% AZCL galactomannan (carob) or any substratefor the assay of endo-1,4-beta-D-mannanase.

Any xylanase, glucanase and/or mannanase assay known in the art can beused to determine if a polypeptide has xylanase, glucanase and/ormannanase activity and is within scope of the invention. For example,reducing sugar assays such as the Nelson-Somogyi method or thedinitrosalicylic acid (DNS) method can be used to assay for the productsugars (and thus, xylanase activity). In one aspect, reactions arecarried out by mixing and incubating a dilution of the enzymepreparation with a known amount of substrate at a buffered pH and settemperature. Xylanase assays are similar to cellulase assays except thata solution of xylan (e.g., oat spelts or birch) is substituted for CMCor filter paper. The DNS assay is easier to use than the Nelson-Somogyiassay. The DNS assay is satisfactory for cellulase activities, but tendsto over estimate xylanase activity. The Somogyi-Nelson procedure is moreaccurate in the determination of reducing sugars, to measure specificactivities and to quantify the total amount of xylanase produced in theoptimized growth conditions, see, e.g., Breuil (1985) Comparison of the3,5-dinitrosalicylic acid and Nelson-Somogyi methods of assaying forreducing sugars and determining cellulase activity, Enzyme Microb.Technol. 7:327-332; Somogyi, M. 1952, Notes on sugar determination, J.Biol. Chem. 195:19-23. The invention incorporates use of any reducingsugar assay, e.g., by Nelson-Somogyi, e.g., based on references Nelson,N. (1944) J. Biol. Chem. 153:375-380, and Somogyi, M. (1952) J. Biol.Chem. 195:19-23.

The polypeptides of the invention include xylanases in an active orinactive form. For example, the polypeptides of the invention includeproproteins before “maturation” or processing of prepro sequences, e.g.,by a proprotein-processing enzyme, such as a proprotein convertase togenerate an “active” mature protein. The polypeptides of the inventioninclude xylanases inactive for other reasons, e.g., before “activation”by a post-translational processing event, e.g., an endo- orexo-peptidase or proteinase action, a phosphorylation event, anamidation, a glycosylation or a sulfation, a dimerization event, and thelike. The polypeptides of the invention include all active forms,including active subsequences, e.g., catalytic domains or active sites,of the xylanase.

Methods for identifying “prepro” domain sequences and signal sequencesare well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136. For example, to identify a prepro sequence, theprotein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. The invention includes polypeptides withheterologous signal sequences and/or prepro sequences. The preprosequence (including a sequence of the invention used as a heterologousprepro domain) can be located on the amino terminal or the carboxyterminal end of the protein. The invention also includes isolated,synthetic or recombinant signal sequences, prepro sequences andcatalytic domains (e.g., “active sites”) comprising sequences of theinvention.

The percent sequence identity can be over the full length of thepolypeptide, or, the identity can be over a region of at least about 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700 or more residues. Polypeptides of the invention can also beshorter than the full length of exemplary polypeptides. In alternativeaspects, the invention provides polypeptides (peptides, fragments)ranging in size between about 5 and the full length of a polypeptide,e.g., an enzyme, such as a xylanase; exemplary sizes being of about 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, ormore residues, e.g., contiguous residues of an exemplary xylanase of theinvention.

Peptides of the invention (e.g., a subsequence of an exemplarypolypeptide of the invention) can be useful as, e.g., labeling probes,antigens, toleragens, motifs, xylanase active sites (e.g., “catalyticdomains”), signal sequences and/or prepro domains.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptide and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these and to naturally occurringor synthetic molecules. “Amino acid” or “amino acid sequence” include anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these, and to naturallyoccurring or synthetic molecules. The term “polypeptide” as used herein,refers to amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres and may contain modified aminoacids other than the 20 gene-encoded amino acids. The polypeptides maybe modified by either natural processes, such as post-translationalprocessing, or by chemical modification techniques that are well knownin the art. Modifications can occur anywhere in the polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also a given polypeptide may have manytypes of modifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphytidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, xylan hydrolase processing, phosphorylation, prenylation,racemization, selenoylation, sulfation and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. (See Creighton,T. E., Proteins—Structure and Molecular Properties 2nd Ed., W.H. Freemanand Company, New York (1993); Posttranslational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, pp. 1-12(1983)). The peptides and polypeptides of the invention also include all“mimetic” and “peptidomimetic” forms, as described in further detail,below.

“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; i.e., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” nucleic acids (includingoligonucleotides), polypeptides or proteins of the invention includethose prepared by any chemical synthesis, e.g., as described, below.Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

“Fragments” or “enzymatically active fragments” as used herein are aportion of an amino acid sequence (encoding a protein) which retains atleast one functional activity of the protein to which it is related.Fragments can have the same or substantially the same amino acidsequence as the naturally occurring protein. “Substantially the same”means that an amino acid sequence is largely, but not entirely, thesame, but retains at least one functional activity of the sequence towhich it is related. In general two amino acid sequences are“substantially the same” or “substantially homologous” if they are atleast about 85% identical. Fragments which have different threedimensional structures as the naturally occurring protein are alsoincluded. An example of this, is a “pro-form” molecule, such as a lowactivity proprotein that can be modified by cleavage to produce a matureenzyme with significantly higher activity.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has axylanase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl) carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl orglutamyl can also be converted to asparaginyl and glutaminyl residues byreaction with ammonium ions. Mimetics of basic amino acids can begenerated by substitution with, e.g., (in addition to lysine andarginine) the amino acids ornithine, citrulline, or (guanidino)-aceticacid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.Nitrile derivative (e.g., containing the CN-moiety in place of COOH) canbe substituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetyllmidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R— or S— form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The invention includes xylanases of the invention with and withoutsignal. The polypeptide comprising a signal sequence of the inventioncan be a xylanase of the invention or another xylanase or another enzymeor other polypeptide.

The invention includes immobilized xylanases, anti-xylanase antibodiesand fragments thereof. The invention provides methods for inhibitingxylanase activity, e.g., using dominant negative mutants oranti-xylanase antibodies of the invention. The invention includesheterocomplexes, e.g., fusion proteins, heterodimers, etc., comprisingthe xylanases of the invention.

Polypeptides of the invention can have a xylanase activity under variousconditions, e.g., extremes in pH and/or temperature, oxidizing agents,and the like. The invention provides methods leading to alternativexylanase preparations with different catalytic efficiencies andstabilities, e.g., towards temperature, oxidizing agents and changingwash conditions. In one aspect, xylanase variants can be produced usingtechniques of site-directed mutagenesis and/or random mutagenesis. Inone aspect, directed evolution can be used to produce a great variety ofxylanase variants with alternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify xylanase modulators, e.g., activators or inhibitors of xylanaseactivity. Briefly, test samples (compounds, broths, extracts, and thelike) are added to xylanase assays to determine their ability to inhibitsubstrate cleavage. Inhibitors identified in this way can be used inindustry and research to reduce or prevent undesired proteolysis. Aswith xylanases, inhibitors can be combined to increase the spectrum ofactivity.

The enzymes of the invention are also useful as research reagents todigest proteins or in protein sequencing. For example, the xylanases maybe used to break polypeptides into smaller fragments for sequencingusing, e.g. an automated sequencer.

The invention also provides methods of discovering new xylanases usingthe nucleic acids, polypeptides and antibodies of the invention. In oneaspect, phagemid libraries are screened for expression-based discoveryof xylanases. In another aspect, lambda phage libraries are screened forexpression-based discovery of xylanases. Screening of the phage orphagemid libraries can allow the detection of toxic clones; improvedaccess to substrate; reduced need for engineering a host, by-passing thepotential for any bias resulting from mass excision of the library; and,faster growth at low clone densities. Screening of phage or phagemidlibraries can be in liquid phase or in solid phase. In one aspect, theinvention provides screening in liquid phase. This gives a greaterflexibility in assay conditions; additional substrate flexibility;higher sensitivity for weak clones; and ease of automation over solidphase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

Another aspect of the invention is an isolated or purified polypeptidecomprising the sequence of one of the invention and sequencessubstantially identical thereto, or fragments comprising at least about5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive aminoacids thereof. As discussed above, such polypeptides may be obtained byinserting a nucleic acid encoding the polypeptide into a vector suchthat the coding sequence is operably linked to a sequence capable ofdriving the expression of the encoded polypeptide in a suitable hostcell. For example, the expression vector may comprise a promoter, aribosome binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression.

Another aspect of the invention is polypeptides or fragments thereofwhich have at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or more than about 95% homology to one of the polypeptides of theinvention and sequences substantially identical thereto, or a fragmentcomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof. Homology may be determined using any ofthe programs described above which aligns the polypeptides or fragmentsbeing compared and determines the extent of amino acid identity orsimilarity between them. It will be appreciated that amino acid“homology” includes conservative amino acid substitutions such as thosedescribed above.

The polypeptides or fragments having homology to one of the polypeptidesof the invention, or a fragment comprising at least about 5, 10, 15, 20,25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof maybe obtained by isolating the nucleic acids encoding them using thetechniques described above.

Alternatively, the homologous polypeptides or fragments may be obtainedthrough biochemical enrichment or purification procedures. The sequenceof potentially homologous polypeptides or fragments may be determined byxylan hydrolase digestion, gel electrophoresis and/or microsequencing.The sequence of the prospective homologous polypeptide or fragment canbe compared to one of the polypeptides of the invention, or a fragmentcomprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or150 consecutive amino acids thereof using any of the programs describedabove.

Another aspect of the invention is an assay for identifying fragments orvariants of The invention, which retain the enzymatic function of thepolypeptides of The invention. For example the fragments or variants ofsaid polypeptides, may be used to catalyze biochemical reactions, whichindicate that the fragment or variant retains the enzymatic activity ofthe polypeptides of the invention.

The assay for determining if fragments of variants retain the enzymaticactivity of the polypeptides of the invention includes the steps of:contacting the polypeptide fragment or variant with a substrate moleculeunder conditions which allow the polypeptide fragment or variant tofunction and detecting either a decrease in the level of substrate or anincrease in the level of the specific reaction product of the reactionbetween the polypeptide and substrate.

The polypeptides of the invention or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof may be used in a variety of applications. For example, thepolypeptides or fragments thereof may be used to catalyze biochemicalreactions. In accordance with one aspect of the invention, there isprovided a process for utilizing the polypeptides of the invention orpolynucleotides encoding such polypeptides for hydrolyzing glycosidiclinkages. In such procedures, a substance containing a glycosidiclinkage (e.g., a starch) is contacted with one of the polypeptides ofThe invention, or sequences substantially identical thereto underconditions which facilitate the hydrolysis of the glycosidic linkage.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds, such assmall molecules. Each biocatalyst is specific for one functional group,or several related functional groups and can react with many startingcompounds containing this functional group.

The biocatalytic reactions produce a population of derivatives from asingle starting compound. These derivatives can be subjected to anotherround of biocatalytic reactions to produce a second population ofderivative compounds. Thousands of variations of the original smallmolecule or compound can be produced with each iteration of biocatalyticderivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies and compounds can be synthesizedand tested free in solution using virtually any type of screening assay.It is important to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods.

In a particular aspect, the invention provides a method for modifyingsmall molecules, comprising contacting a polypeptide encoded by apolynucleotide described herein or enzymatically active fragmentsthereof with a small molecule to produce a modified small molecule. Alibrary of modified small molecules is tested to determine if a modifiedsmall molecule is present within the library which exhibits a desiredactivity. A specific biocatalytic reaction which produces the modifiedsmall molecule of desired activity is identified by systematicallyeliminating each of the biocatalytic reactions used to produce a portionof the library and then testing the small molecules produced in theportion of the library for the presence or absence of the modified smallmolecule with the desired activity. The specific biocatalytic reactionswhich produce the modified small molecule of desired activity is in oneaspect (optionally) repeated. The biocatalytic reactions are conductedwith a group of biocatalysts that react with distinct structuralmoieties found within the structure of a small molecule, eachbiocatalyst is specific for one structural moiety or a group of relatedstructural moieties; and each biocatalyst reacts with many differentsmall molecules which contain the distinct structural moiety.

Xylanase Signal Sequences, Prepro and Catalytic Domains

The invention provides xylanase signal sequences (e.g., signal peptides(SPs)), prepro domains and catalytic domains (CDs). The SPs, preprodomains and/or CDs of the invention can be isolated, synthetic orrecombinant peptides or can be part of a fusion protein, e.g., as aheterologous domain in a chimeric protein. The invention providesnucleic acids encoding these catalytic domains (CDs), prepro domains andsignal sequences (SPs, e.g., a peptide having a sequencecomprising/consisting of amino terminal residues of a polypeptide of theinvention). In one aspect, the invention provides a signal sequencecomprising a peptide comprising/consisting of a sequence as set forth inresidues 1 to 12, 1 to 13, 1 to 14,1 to 15,1 to 16, 1 to 17,1 to 18,1 to19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26, 1 to27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34, 1 to35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42, 1 to43, 1 to 44, 1 to 45, 1 to 46, 1 to 47, 1 to 48, 1 to 49 or 1 to 50, ofa polypeptide of the invention.

The xylanase signal sequences (SPs) and/or prepro sequences of theinvention can be isolated peptides, or, sequences joined to anotherxylanase or a non-xylanase polypeptide, e.g., as a fusion (chimeric)protein. In one aspect, the invention provides polypeptides comprisingxylanase signal sequences of the invention. In one aspect, polypeptidescomprising xylanase signal sequences SPs and/or prepro of the inventioncomprise sequences heterologous to a xylanase of the invention (e.g., afusion protein comprising an SP and/or prepro of the invention andsequences from another xylanase or a non-xylanase protein). In oneaspect, the invention provides xylanases of the invention withheterologous SPs and/or prepro sequences, e.g., sequences with a yeastsignal sequence. A xylanase of the invention can comprise a heterologousSP and/or prepro in a vector, e.g., a pPIC series vector (Invitrogen,Carlsbad, Calif.).

In one aspect, SPs and/or prepro sequences of the invention areidentified following identification of novel xylanase polypeptides. Thepathways by which proteins are sorted and transported to their propercellular location are often referred to as protein targeting pathways.One of the most important elements in all of these targeting systems isa short amino acid sequence at the amino terminus of a newly synthesizedpolypeptide called the signal sequence. This signal sequence directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. Mostlysosomal, membrane, or secreted proteins have an amino-terminal signalsequence that marks them for translocation into the lumen of theendoplasmic reticulum. More than 100 signal sequences for proteins inthis group have been determined. The signal sequences can vary in lengthfrom between about 11 to 41, or between about 13 to 36 amino acidresidues. Various methods of recognition of signal sequences are knownto those of skill in the art. For example, in one aspect, novel xylanasesignal peptides are identified by a method referred to as SignalP.SignalP uses a combined neural network which recognizes both signalpeptides and their cleavage sites; see, e.g., Nielsen (1997)“Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering 10:1-6.

It should be understood that in some aspects xylanases of the inventionmay not have SPs and/or prepro sequences, or “domains.” In one aspect,the invention provides the xylanases of the invention lacking all orpart of an SP and/or a prepro domain. In one aspect, the inventionprovides a nucleic acid sequence encoding a signal sequence (SP) and/orprepro from one xylanase operably linked to a nucleic acid sequence of adifferent xylanase or, in one aspect (optionally), a signal sequence(SPs) and/or prepro domain from a non-xylanase protein may be desired.

The invention also provides isolated, synthetic or recombinantpolypeptides comprising signal sequences (SPs), prepro domain and/orcatalytic domains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toa xylanase) with an SP, prepro domain and/or CD. The sequence to whichthe SP, prepro domain and/or CD are not naturally associated can be onthe SP's, prepro domain and/or CD's amino terminal end, carboxy terminalend, and/or on both ends of the SP and/or CD. In one aspect, theinvention provides an isolated, synthetic or recombinant polypeptidecomprising (or consisting of) a polypeptide comprising a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention withthe proviso that it is not associated with any sequence to which it isnaturally associated (e.g., a xylanase sequence). Similarly in oneaspect, the invention provides isolated, synthetic or recombinantnucleic acids encoding these polypeptides. Thus, in one aspect, theisolated, synthetic or recombinant nucleic acid of the inventioncomprises coding sequence for a signal sequence (SP), prepro domainand/or catalytic domain (CD) of the invention and a heterologoussequence (i.e., a sequence not naturally associated with the a signalsequence (SP), prepro domain and/or catalytic domain (CD) of theinvention). The heterologous sequence can be on the 3′ terminal end, 5′terminal end, and/or on both ends of the SP, prepro domain and/or CDcoding sequence.

Hybrid (Chimeric) Xylanases and Peptide Libraries

In one aspect, the invention provides hybrid xylanases and fusionproteins, including peptide libraries, comprising sequences of theinvention. The peptide libraries of the invention can be used to isolatepeptide modulators (e.g., activators or inhibitors) of targets, such asxylanase substrates, receptors, enzymes. The peptide libraries of theinvention can be used to identify formal binding partners of targets,such as ligands, e.g., cytokines, hormones and the like. In one aspect,the invention provides chimeric proteins comprising a signal sequence(SP), prepro domain and/or catalytic domain (CD) of the invention or acombination thereof and a heterologous sequence (see above).

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of xylanases of the invention and other peptides, includingknown and random peptides. They can be fused in such a manner that thestructure of the xylanases is not significantly perturbed and thepeptide is metabolically or structurally conformationally stabilized.This allows the creation of a peptide library that is easily monitoredboth for its presence within cells and its quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of a xylanase sequence. In one aspect, the variants of theinvention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed xylanase variants screened for the optimal combinationof desired activity. Techniques for making substitution mutations atpredetermined sites in DNA having a known sequence are well known, asdiscussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using, e.g., assays ofxylan hydrolysis. In alternative aspects, amino acid substitutions canbe single residues; insertions can be on the order of from about 1 to 20amino acids, although considerably larger insertions can be done.Deletions can range from about 1 to about 20, 30, 40, 50, 60, 70residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

The invention provides xylanases where the structure of the polypeptidebackbone, the secondary or the tertiary structure, e.g., analpha-helical or beta-sheet structure, has been modified. In one aspect,the charge or hydrophobicity has been modified. In one aspect, the bulkof a side chain has been modified. Substantial changes in function orimmunological identity are made by selecting substitutions that are lessconservative. For example, substitutions can be made which moresignificantly affect: the structure of the polypeptide backbone in thearea of the alteration, for example a alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e. xylanase activity) although variants can be selected tomodify the characteristics of the xylanases as needed.

In one aspect, xylanases of the invention comprise epitopes orpurification tags, signal sequences or other fusion sequences, etc. Inone aspect, the xylanases of the invention can be fused to a randompeptide to form a fusion polypeptide. By “fused” or “operably linked”herein is meant that the random peptide and the xylanase are linkedtogether, in such a manner as to minimize the disruption to thestability of the xylanase structure, e.g., it retains xylanase activity.The fusion polypeptide (or fusion polynucleotide encoding the fusionpolypeptide) can comprise further components as well, including multiplepeptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

Xylanases are multidomain enzymes that consist in one aspect(optionally) of a signal peptide, a carbohydrate binding module, axylanase catalytic domain, a linker and/or another catalytic domain.

The invention provides a means for generating chimeric polypeptideswhich may encode biologically active hybrid polypeptides (e.g., hybridxylanases). In one aspect, the original polynucleotides encodebiologically active polypeptides. The method of the invention producesnew hybrid polypeptides by utilizing cellular processes which integratethe sequence of the original polynucleotides such that the resultinghybrid polynucleotide encodes a polypeptide demonstrating activitiesderived from the original biologically active polypeptides. For example,the original polynucleotides may encode a particular enzyme fromdifferent microorganisms. An enzyme encoded by a first polynucleotidefrom one organism or variant may, for example, function effectivelyunder a particular environmental condition, e.g. high salinity. Anenzyme encoded by a second polynucleotide from a different organism orvariant may function effectively under a different environmentalcondition, such as extremely high temperatures. A hybrid polynucleotidecontaining sequences from the first and second original polynucleotidesmay encode an enzyme which exhibits characteristics of both enzymesencoded by the original polynucleotides. Thus, the enzyme encoded by thehybrid polynucleotide may function effectively under environmentalconditions shared by each of the enzymes encoded by the first and secondpolynucleotides, e.g., high salinity and extreme temperatures.

Enzymes encoded by the polynucleotides of the invention include, but arenot limited to, hydrolases, such as xylanases. Glycosidase hydrolaseswere first classified into families in 1991, see, e.g., Henrissat (1991)Biochem. J. 280:309-316. Since then, the classifications have beencontinually updated, see, e.g., Henrissat (1993) Biochem. J.293:781-788; Henrissat (1996) Biochem. J. 316:695-696; Henrissat (2000)Plant Physiology 124:1515-1519. There are 87 identified families ofglycosidase hydrolases. In one aspect, the xylanases of the inventionmay be categorized in families 8, 10, 11, 26 and 30. In one aspect, theinvention also provides xylanase-encoding nucleic acids with a commonnovelty in that they are derived from a common family, e.g., 11.

A hybrid polypeptide resulting from the method of the invention mayexhibit specialized enzyme activity not displayed in the originalenzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding hydrolase activities, theresulting hybrid polypeptide encoded by a hybrid polynucleotide can bescreened for specialized hydrolase activities obtained from each of theoriginal enzymes, i.e. the type of bond on which the hydrolase acts andthe temperature at which the hydrolase functions. Thus, for example, thehydrolase may be screened to ascertain those chemical functionalitieswhich distinguish the hybrid hydrolase from the original hydrolases,such as: (a) amide (peptide bonds), i.e., xylanases; (b) ester bonds,i.e., esterases and lipases; (c) acetals, i.e., glycosidases and, forexample, the temperature, pH or salt concentration at which the hybridpolypeptide functions.

Sources of the original polynucleotides may be isolated from individualorganisms (“isolates”), collections of organisms that have been grown indefined media (“enrichment cultures”), or, uncultivated organisms(“environmental samples”). The use of a culture-independent approach toderive polynucleotides encoding novel bioactivities from environmentalsamples is most preferable since it allows one to access untappedresources of biodiversity.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

Additionally, subcloning may be performed to further isolate sequencesof interest. In subcloning, a portion of DNA is amplified, digested,generally by restriction enzymes, to cut out the desired sequence, thedesired sequence is ligated into a recipient vector and is amplified. Ateach step in subcloning, the portion is examined for the activity ofinterest, in order to ensure that DNA that encodes the structuralprotein has not been excluded. The insert may be purified at any step ofthe subcloning, for example, by gel electrophoresis prior to ligationinto a vector or where cells containing the recipient vector and cellsnot containing the recipient vector are placed on selective mediacontaining, for example, an antibiotic, which will kill the cells notcontaining the recipient vector. Specific methods of subcloning cDNAinserts into vectors are well-known in the art (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989)). In another aspect, the enzymes of theinvention are subclones. Such subclones may differ from the parent cloneby, for example, length, a mutation, a tag or a label.

It should be understood that some of the xylanases of the invention mayor may not contain signal sequences. It may be desirable to include anucleic acid sequence encoding a signal sequence from one xylanaseoperably linked to a nucleic acid sequence of a different xylanase or,in one aspect (optionally), a signal sequence from a non-xylanaseprotein may be desired.

The microorganisms from which the polynucleotide may be prepared includeprokaryotic microorganisms, such as Eubacteria and Archaebacteria andlower eukaryotic microorganisms such as fungi, some algae and protozoa.Polynucleotides may be isolated from environmental samples in which casethe nucleic acid may be recovered without culturing of an organism orrecovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.Polynucleotides encoding enzymes isolated from extremophilicmicroorganisms can be used. Such enzymes may function at temperaturesabove 100° C. in terrestrial hot springs and deep sea thermal vents, attemperatures below 0° C. in arctic waters, in the saturated saltenvironment of the Dead Sea, at pH values around 0 in coal deposits andgeothermal sulfur-rich springs, or at pH values greater than 11 insewage sludge. For example, several esterases and lipases cloned andexpressed from extremophilic organisms show high activity throughout awide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are preferably already in avector which includes appropriate control sequences. The host cell canbe a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or preferably, the host cell canbe a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986).

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; and plant cells. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981) and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In another aspect, it is envisioned the method of the present inventioncan be used to generate novel polynucleotides encoding biochemicalpathways from one or more operons or gene clusters or portions thereof.For example, bacteria and many eukaryotes have a coordinated mechanismfor regulating genes whose products are involved in related processes.The genes are clustered, in structures referred to as “gene clusters,”on a single chromosome and are transcribed together under the control ofa single regulatory sequence, including a single promoter whichinitiates transcription of the entire cluster. Thus, a gene cluster is agroup of adjacent genes that are either identical or related, usually asto their function. An example of a biochemical pathway encoded by geneclusters are polyketides.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Oneaspect of the invention is to use cloning vectors, referred to as“fosmids” or bacterial artificial chromosome (BAC) vectors. These arederived from E. coli f-factor which is able to stably integrate largesegments of genomic DNA. When integrated with DNA from a mixeduncultured environmental sample, this makes it possible to achieve largegenomic fragments in the form of a stable “environmental DNA library.”Another type of vector for use in the present invention is a cosmidvector. Cosmid vectors were originally designed to clone and propagatelarge segments of genomic DNA. Cloning into cosmid vectors is describedin detail in Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press (1989). Once ligated intoan appropriate vector, two or more vectors containing differentpolyketide synthase gene clusters can be introduced into a suitable hostcell. Regions of partial sequence homology shared by the gene clusterswill promote processes which result in sequence reorganization resultingin a hybrid gene cluster. The novel hybrid gene cluster can then bescreened for enhanced activities not found in the original geneclusters.

Therefore, in a one aspect, the invention relates to a method forproducing a biologically active hybrid polypeptide and screening such apolypeptide for enhanced activity by:

1) introducing at least a first polynucleotide in operable linkage and asecond polynucleotide in operable linkage, the at least firstpolynucleotide and second polynucleotide sharing at least one region ofpartial sequence homology, into a suitable host cell;

2) growing the host cell under conditions which promote sequencereorganization resulting in a hybrid polynucleotide in operable linkage;

3) expressing a hybrid polypeptide encoded by the hybrid polynucleotide;

4) screening the hybrid polypeptide under conditions which promoteidentification of enhanced biological activity; and

5) isolating the a polynucleotide encoding the hybrid polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

Screening Methodologies and “On-line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides forxylanase activity (e.g., assays such as hydrolysis of casein inzymograms, the release of fluorescence from gelatin, or the release ofp-nitroanalide from various small peptide substrates), to screencompounds as potential modulators, e.g., activators or inhibitors, of axylanase activity, for antibodies that bind to a polypeptide of theinvention, for nucleic acids that hybridize to a nucleic acid of theinvention, to screen for cells expressing a polypeptide of the inventionand the like. In addition to the array formats described in detail belowfor screening samples, alternative formats can also be used to practicethe methods of the invention. Such formats include, for example, massspectrometers, chromatographs, e.g., high-throughput HPLC and otherforms of liquid chromatography, and smaller formats, such as 1536-wellplates, 384—well plates and so on. High throughput screening apparatuscan be adapted and used to practice the methods of the invention, see,e.g., U.S. Patent Application No. 20020001809.

Capillary Arrays

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. Capillary arrays, suchas the GIGAMATRIX™, Diversa Corporation, San Diego, Calif.; and arraysdescribed in, e.g., U.S. Patent Application No. 20020080350 A1; WO0231203 A; WO 0244336 A, provide an alternative apparatus for holdingand screening samples. In one aspect, the capillary array includes aplurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The lumen may be cylindrical, square, hexagonal orany other geometric shape so long as the walls form a lumen forretention of a liquid or sample. The capillaries of the capillary arraycan be held together in close proximity to form a planar structure. Thecapillaries can be bound together, by being fused (e.g., where thecapillaries are made of glass), glued, bonded, or clamped side-by-side.Additionally, the capillary array can include interstitial materialdisposed between adjacent capillaries in the array, thereby forming asolid planar device containing a plurality of through-holes.

A capillary array can be formed of any number of individual capillaries,for example, a range from 100 to 4,000,000 capillaries. Further, acapillary array having about 100,000 or more individual capillaries canbe formed into the standard size and shape of a Microtiter® plate forfitment into standard laboratory equipment. The lumens are filledmanually or automatically using either capillary action ormicroinjection using a thin needle. Samples of interest may subsequentlybe removed from individual capillaries for further analysis orcharacterization. For example, a thin, needle-like probe is positionedin fluid communication with a selected capillary to either add orwithdraw material from the lumen.

In a single-pot screening assay, the assay components are mixed yieldinga solution of interest, prior to insertion into the capillary array. Thelumen is filled by capillary action when at least a portion of the arrayis immersed into a solution of interest. Chemical or biologicalreactions and/or activity in each capillary are monitored for detectableevents. A detectable event is often referred to as a “hit”, which canusually be distinguished from “non-hit” producing capillaries by opticaldetection. Thus, capillary arrays allow for massively parallel detectionof “hits”.

In a multi-pot screening assay, a polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component, which is introducedinto at least a portion of a capillary of a capillary array. An airbubble can then be introduced into the capillary behind the firstcomponent. A second component can then be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. The first and second components can then be mixed byapplying hydrostatic pressure to both sides of the capillary array tocollapse the bubble. The capillary array is then monitored for adetectable event resulting from reaction or non-reaction of the twocomponents.

In a binding screening assay, a sample of interest can be introduced asa first liquid labeled with a detectable particle into a capillary of acapillary array, wherein the lumen of the capillary is coated with abinding material for binding the detectable particle to the lumen. Thefirst liquid may then be removed from the capillary tube, wherein thebound detectable particle is maintained within the capillary, and asecond liquid may be introduced into the capillary tube. The capillaryis then monitored for a detectable event resulting from reaction ornon-reaction of the particle with the second liquid.

Arrays, or “Biochips”

Nucleic acids and/or polypeptides of the invention can be immobilized toor applied to an array, e.g., a “biochip”. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. For example, in one aspect of the invention, a monitoredparameter is transcript expression of a xylanase gene. One or more, or,all the transcripts of a cell can be measured by hybridization of asample comprising transcripts of the cell, or, nucleic acidsrepresentative of or complementary to transcripts of a cell, byhybridization to immobilized nucleic acids on an array, or “biochip.” Byusing an “array” of nucleic acids on a microchip, some or all of thetranscripts of a cell can be simultaneously quantified. Alternatively,arrays comprising genomic nucleic acid can also be used to determine thegenotype of a newly engineered strain made by the methods of theinvention. Polypeptide arrays” can also be used to simultaneouslyquantify a plurality of proteins. The present invention can be practicedwith any known “array,” also referred to as a “microarray” or “nucleicacid array” or “polypeptide array” or “antibody array” or “biochip,” orvariation thereof. Arrays are generically a plurality of “spots” or“target elements,” each target element comprising a defined amount ofone or more biological molecules, e.g., oligonucleotides, immobilizedonto a defined area of a substrate surface for specific binding to asample molecule, e.g., mRNA transcripts.

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171—R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to a xylanase of the invention. These antibodiescan be used to isolate, identify or quantify the xylanases of theinvention or related polypeptides. These antibodies can be used toisolate other polypeptides within the scope the invention or otherrelated xylanases. The antibodies can be designed to bind to an activesite of a xylanase. Thus, the invention provides methods of inhibitingxylanases using the antibodies of the invention (see discussion aboveregarding applications for anti-xylanase compositions of the invention).The invention provides fragments of the enzymes of the invention,including immunogenic fragments of a polypeptide of the invention. Theinvention provides compositions comprising a polypeptide or peptide ofthe invention and adjuvants or carriers and the like.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y. (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides of The invention or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof, may also be used to generate antibodies which bind specificallyto the polypeptides or fragments. The resulting antibodies may be usedin immunoaffinity chromatography procedures to isolate or purify thepolypeptide or to determine whether the polypeptide is present in abiological sample. In such procedures, a protein preparation, such as anextract, or a biological sample is contacted with an antibody capable ofspecifically binding to one of the polypeptides of The invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of The invention,or fragment thereof. After a wash to remove non-specifically boundproteins, the specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays and Western Blots.

Polyclonal antibodies generated against the polypeptides of Theinvention, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,40, 50, 75, 100, or 150 or more consecutive amino acids thereof can beobtained by direct injection of the polypeptides into an animal or byadministering the polypeptides to an animal, for example, a nonhuman.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72, 1983) and theEBV-hybridoma technique (Cole, et al., 1985, in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies tothe polypeptides of The invention, or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof. Alternatively, transgenic mice may be used to express humanizedantibodies to these polypeptides or fragments thereof.

Antibodies generated against the polypeptides of The invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof may be used in screening forsimilar polypeptides from other organisms and samples. In suchtechniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding. One such screening assay is described in “Methods forMeasuring Cellulase Activities”, Methods in Enzymology, Vol 160, pp.87-116.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., xylanases) and/or antibodies of theinvention. The kits also can contain instructional material teaching themethodologies and industrial, research, medical, pharmaceutical, foodand feed and food and feed supplement processing and other applicationsand processes of the invention, as described herein.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a new or modified xylanase activity, by modifying thegenetic composition of the cell. The genetic composition can be modifiedby addition to the cell of a nucleic acid of the invention, e.g., acoding sequence for an enzyme of the invention. See, e.g., WO0229032;WO0196551.

To detect the new phenotype, at least one metabolic parameter of amodified cell is monitored in the cell in a “real time” or “on-line”time frame. In one aspect, a plurality of cells, such as a cell culture,is monitored in “real time” or “on-line.” In one aspect, a plurality ofmetabolic parameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using the xylanases of the invention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., axylanase message) or generating new (e.g., xylanase) transcripts in acell. This increased or decreased expression can be traced by testingfor the presence of a xylanase of the invention or by xylanase activityassays. mRNA transcripts, or messages, also can be detected andquantified by any method known in the art, including, e.g., Northernblots, quantitative amplification reactions, hybridization to arrays,and the like. Quantitative amplification reactions include, e.g.,quantitative PCR, including, e.g., quantitative reverse transcriptionpolymerase chain reaction, or RT-PCR; quantitative real time RT-PCR, or“real-time kinetic RT-PCR” (see, e.g., Kreuzer (2001) Br. J. Haematol.114:313-318; Xia (2001) Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., axylanase) or generating new polypeptides in a cell. This increased ordecreased expression can be traced by determining the amount of xylanasepresent or by xylanase activity assays. Polypeptides, peptides and aminoacids also can be detected and quantified by any method known in theart, including, e.g., nuclear magnetic resonance (NMR),spectrophotometry, radiography (protein radiolabeling), electrophoresis,capillary electrophoresis, high performance liquid chromatography(HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography,various immunological methods, e.g. immunoprecipitation,immunodiffusion, immuno-electrophoresis, radioimmunoassays (RIAs),enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays,gel electrophoresis (e.g., SDS-PAGE), staining with antibodies,fluorescent activated cell sorter (FACS), pyrolysis mass spectrometry,Fourier-Transform Infrared Spectrometry, Raman spectrometry, GC-MS, andLC-Electrospray and cap-LC-tandem-electrospray mass spectrometries, andthe like. Novel bioactivities can also be screened using methods, orvariations thereof, described in U.S. Pat. No. 6,057,103. Furthermore,as discussed below in detail, one or more, or, all the polypeptides of acell can be measured using a protein array.

Industrial, Energy, Pharmaceutical, Medical, Food Processing and OtherApplications

Polypeptides of the invention can be used in any industrial,agricultural, food and feed and food and feed supplement processing,pharmaceutical, medical, research (laboratory) or other process. Theinvention provides industrial processes using enzymes of the invention,e.g., in the pharmaceutical or nutrient (diet) supplement industry, theenergy industry (e.g., to make “clean” biofuels), in the food and feedindustries, e.g., in methods for making food and feed products and foodand feed additives. In one aspect, the invention provides processesusing enzymes of the invention in the medical industry, e.g., to makepharmaceuticals or dietary aids or supplements, or food supplements andadditives. In addition, the invention provides methods for using theenzymes of the invention in biofuel production, including, e.g., abioalcohol such as bioethanol, biomethanol, biobutanol or biopropanol,thus comprising a “clean” fuel production. Enzymes of the invention canbe added to industrial processes continuously, in batches or byfed-batch methods. In another aspect, enzymes of the invention can berecycled in the industrial processes, thereby lowering enzymerequirements.

For example, xylanases can be used in the biobleaching and treatment ofchemical pulps, for example, as described in U.S. Pat. No. 5,202,249; orfor biobleaching and treatment of wood or paper pulps, for example, asdescribed in U.S. Pat. Nos. 5,179,021, 5,116,746, 5,407,827, 5,405,769,5,395,765, 5,369,024, 5,457,045, 5,434,071, 5,498,534, 5,591,304,5,645,686, 5,725,732, 5,759,840, 5,834,301, 5,871,730 and 6,057,438; orfor reducing lignin in wood and modifying wood, for example, asdescribed in U.S. Pat. No. 5,486,468 and/or 5,770,012; or for use asflour, dough and bread improvers, for example, as described in U.S. Pat.Nos. 5,108,765 and/or 5,306,633; or for use as feed additives and/orsupplements, for example, as described in U.S. Pat. Nos. 5,432,074;5,429,828; 5,612,055; 5,720,971; 5,981,233; 5,948,667; 6,099,844;6,132,727 and/or 6,132,716; or in manufacturing cellulose solutions, forexample, as described in U.S. Pat. No. 5,760,211; or in detergentcompositions; or used for fruit, vegetables and/or mud and claycompounds, for example, as described in U.S. Pat. No. 5,786,316.Xylanases of this invention also can be used in hydrolysis ofhemicellulose, for example, as described in U.S. Pat. No. 4,725,544.

The xylanase enzymes of the invention can be highly selective catalysts.They can catalyze reactions with exquisite stereo-, regio- andchemo-selectivities that are unparalleled in conventional syntheticchemistry. Moreover, enzymes are remarkably versatile. The xylanaseenzymes of the invention can be tailored to function in organicsolvents, operate at extreme pHs (for example, high pHs and low pHs)extreme temperatures (for example, high temperatures and lowtemperatures), extreme salinity levels (for example, high salinity andlow salinity) and catalyze reactions with compounds that arestructurally unrelated to their natural, physiological substrates.

Wood, paper and pulp treatments

The xylanases of the invention can be used in any wood, wood product,wood waste or by-product, paper, paper product, paper or wood pulp,Kraft pulp, or wood or paper recycling treatment or industrial process,e.g., any wood, wood pulp, paper waste, paper or pulp treatment or woodor paper deinking process. In one aspect, xylanases of the invention canbe used to treat/pretreat paper pulp, or recycled paper or paper pulp,and the like. In one aspect, enzyme(s) of the invention are used toincrease the “brightness” of the paper via their use intreating/pretreating paper pulp, or recycled paper or paper pulp, andthe like. The higher the grade of paper, the greater the brightness;paper brightness can impact the scan capability of optical scanningequipment; thus, the enzymes and processes of the invention can be usedto make high grade, “bright” paper for, e.g., use in optical scanningequipment, including inkjet, laser and photo printing quality paper.

For example, the enzymes of the invention can be used in any industrialprocess using xylanases known in the art, e.g., treating waste paper, asdescribed in, e.g., U.S. Pat. No. 6,767,728 or 6,426,200; seasoningwood, e.g., for applications in the food industry, as described in,e.g., U.S. Pat. No. 6,623,953; for the production of xylose from apaper-grade hardwood pulp, as described in, e.g., U.S. Pat. No.6,512,110; treating fibrous lignocellulosic raw material with a xylanasein an aqueous medium as described in, e.g., U.S. Pat. No. 6,287,708;dissolving pulp from cellulosic fiber, as described in, e.g., U.S. Pat.No. 6,254,722; deinking and decolorizing a printed paper or removingcolor from wood pulp, as described in, e.g., U.S. Pat. No. 6,241,849,5,834,301 or 5,582,681; bleaching a chemical paper pulp orlignocellulose pulp using a xylanase, as described in, e.g., U.S. Pat.No. 5,645,686 or 5,618,386; for treating wood pulp that includesincompletely washed brownstock, as described in, e.g., U.S. Pat. No.5,591,304; purifying and delignifying a waste lignocellulosic material,as described in, e.g., in U.S. Pat. No. 5,503,709; manufacturing paperor cardboard from recycled cellulose fibers, as described in, e.g., inU.S. Pat. No. 5,110,412; debarking of logs, as described in, e.g., inU.S. Pat. No. 5,103,883; producing fluff pulp with improved shreddingproperties, as described in, e.g., in U.S. Pat. No. 5,068,009, and thelike. The xylanases of the invention can be used to process or treat anycellulosic material, e.g., fibers from wood, cotton, hemp, flax orlinen.

In one aspect, the invention provides wood, wood pulp, paper, paperpulp, paper waste or wood or paper recycling treatment processes using axylanase of the invention. In one aspect, the xylanase of the inventionis applicable both in reduction of the need for a chemical decoloring(e.g., bleaching) agent, such as chlorine dioxide, and in high alkalineand high temperature environments. Most lignin is solubilized in thecooking stage of pulping process. The residual lignin is removed fromthe pulp in the bleaching process. In one aspect, xylanase bleaching ofpulp (e.g., using an enzyme of the invention) is based on the partialhydrolysis of xylan, which is the main component of the hemicellulose.The enzymatic action (e.g., of an enzyme of the invention) releaseshemicellulose-bound lignin and increases the extractability of ligninfrom the pulp in the subsequent bleaching process, e.g. using chlorineand oxygen chemicals. In one aspect, xylanases of the invention can beused to increase the final brightness of the pulp at a fixed level ofbleaching chemicals. In another aspect, xylanases of the invention canbe used to decrease the kappa number of the pulp.

The invention provides wood, wood pulp, paper, paper pulp, paper wasteor wood or paper recycling treatment processes (methods) using axylanase of the invention where the treatment time (the amount of timethe xylanase is in contact with the pulp, paper, wood, etc.) and/orretention time can be anywhere from between about 1 minute to 12 hours,or between about 5 minutes to 1 hour, or between about 15 to 30 minutes;or the treatment and/or retention time can be any time up to about 0.1,0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or morehours.

In one aspect, the xylanase of the invention is a thermostable alkalineendoxylanase which in one aspect can effect a greater than 25% reductionin the chlorine dioxide requirement of kraft pulp with a less than 0.5%pulp yield loss. In one aspect, boundary parameters are pH 10, 65-85° C.and treatment time of less than 60 minutes at an enzyme loading of lessthan 0.001 wt %; in alternative aspects the treatment and/or retentiontime is less than about 0.1, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or 12.

A pool of xylanases may be tested for the ability to hydrolyzedye-labeled xylan at, for example, pH 10 and 60° C. The enzymes thattest positive under these conditions may then be evaluated at, forexample pH 10 and 70° C. Alternatively, enzymes may be tested at pH 8and pH 10 at 70° C. In discovery of xylanases desirable in the pulp andpaper industry libraries from high temperature or highly alkalineenvironments were targeted. Specifically, these libraries were screenedfor enzymes functioning at alkaline pH and a temperature ofapproximately 45° C. In another aspect, the xylanases of the inventionare useful in the pulp and paper industry in degradation of alignin-hemicellulose linkage, in order to release the lignin.

Enzymes of the invention can be used for deinking printed wastepaper,such as newspaper, or for deinking noncontact-printed wastepaper, e.g.,xerographic and laser-printed paper, and mixtures of contact andnoncontact-printed wastepaper, as described in U.S. Pat. No. 6,767,728or 6,426,200; Neo (1986) J. Wood Chem. Tech. 6(2):147. Enzymes of theinvention can be used in processes for the production of xylose from apaper-grade hardwood pulp by extracting xylan contained in pulp into aliquid phase, subjecting the xylan contained in the obtained liquidphase to conditions sufficient to hydrolyze xylan to xylose, andrecovering the xylose, where the extracting step includes at least onetreatment of an aqueous suspension of pulp or an alkali-soluble materiala xylanase enzyme, as described in, e.g., U.S. Pat. No. 6,512,110.Enzymes of the invention can be used in processes for dissolving pulpfrom cellulosic fibers such as recycled paper products made fromhardwood fiber, a mixture of hardwood fiber and softwood fiber, wastepaper, e.g., from unprinted envelopes, de-inked envelopes, unprintedledger paper, de-inked ledger paper, and the like, as described in,e.g., U.S. Pat. No. 6,254,722.

In another aspect of the invention, the xylanases of the invention canalso be used in any wood, wood product, paper, paper product, paper orwood pulp, Kraft pulp, or wood or paper recycling treatment orindustrial process, e.g., any wood, wood pulp, paper waste, paper orpulp treatment or wood or paper deinking process as an antimicrobial ormicrobial repellent. Alternatively, the xylanases of the invention canbe part of a wood, wood product, paper, paper product, paper or woodpulp, Kraft pulp, or recycled paper composition, and/or a compositioncomprising one or more wood, wood product, paper, paper product, paperor wood pulp, Kraft pulp, or recycled paper compositions, wherein thexylanases of the invention act as an antimicrobial or microbialrepellent in the composition.

Treating Fibers and Textiles

The invention provides methods of treating fibers and fabrics using oneor more xylanases of the invention. The xylanases can be used in anyfiber- or fabric-treating method, which are well known in the art, see,e.g., U.S. Pat. Nos. 6,261,828; 6,077,316; 6,024,766; 6,021,536;6,017,751; 5,980,581; US Patent Publication No. 20020142438 A1. Forexample, xylanases of the invention can be used in fiber and/or fabricdesizing. In one aspect, the feel and appearance of a fabric is improvedby a method comprising contacting the fabric with a xylanase of theinvention in a solution. In one aspect, the fabric is treated with thesolution under pressure. For example, xylanases of the invention can beused in the removal of stains.

The xylanases of the invention can be used to treat any cellulosicmaterial, including fibers (e.g., fibers from cotton, hemp, flax orlinen), sewn and unsewn fabrics, e.g., knits, wovens, denims, yarns, andtoweling, made from cotton, cotton blends or natural or manmadecellulosics (e.g. originating from xylan-containing cellulose fiberssuch as from wood pulp) or blends thereof. Examples of blends are blendsof cotton or rayon/viscose with one or more companion material such aswool, synthetic fibers (e.g. polyamide fibers, acrylic fibers, polyesterfibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell).

The textile treating processes of the invention (using xylanases of theinvention) can be used in conjunction with other textile treatments,e.g., scouring and bleaching. Scouring is the removal of non-cellulosicmaterial from the cotton fiber, e.g., the cuticle (mainly consisting ofwaxes) and primary cell wall (mainly consisting of pectin, protein andxyloglucan). A proper wax removal is necessary for obtaining a highwettability. This is needed for dyeing. Removal of the primary cellwalls by the processes of the invention improves wax removal and ensuresa more even dyeing. Treating textiles with the processes of theinvention can improve whiteness in the bleaching process. The mainchemical used in scouring is sodium, hydroxide in high concentrationsand at high temperatures. Bleaching comprises oxidizing the textile.Bleaching typically involves use of hydrogen peroxide as the oxidizingagent in order to obtain either a fully bleached (white) fabric or toensure a clean shade of the dye.

The invention also provides alkaline xylanases (xylanases active underalkaline conditions). These have wide-ranging applications in textileprocessing, degumming of plant fibers (e.g., plant bast fibers),treatment of pectic wastewaters, paper-making, and coffee and teafermentations. See, e.g., Hoondal (2002) Applied Microbiology andBiotechnology 59:409-418.

In another aspect of the invention, the xylanases of the invention canalso be used in any fiber- and/or fabric-treating process as anantimicrobial or microbial repellent. Alternatively, the xylanases ofthe invention can be part of a fiber- and/or fabric-composition, wherethe xylanases of the invention act as an antimicrobial or microbialrepellent in the fiber and/or fabric.

Detergent, Disinfectant and Cleaning Compositions

the invention provides detergent, disinfectant or cleanser (cleaning orcleansing) compositions comprising one or more polypeptides (e.g.,xylanases) of the invention, and methods of making and using thesecompositions. The invention incorporates all methods of making and usingdetergent, disinfectant or cleanser compositions, see, e.g., U.S. Pat.No. 6,413,928; 6,399,561; 6,365,561; 6,380,147. The detergent,disinfectant or cleanser compositions can be a one and two part aqueouscomposition, a non-aqueous liquid composition, a cast solid, a granularform, a particulate form, a compressed tablet, a gel and/or a paste anda slurry form. The xylanases of the invention can also be used as adetergent, disinfectant or cleanser additive product in a solid or aliquid form. Such additive products are intended to supplement or boostthe performance of conventional detergent compositions and can be addedat any stage of the cleaning process.

The actual active enzyme content depends upon the method of manufactureof a detergent, disinfectant or cleanser composition and is notcritical, assuming the detergent solution has the desired enzymaticactivity. In one aspect, the amount of xylanase present in the finalsolution ranges from about 0.001 mg to 0.5 mg per gram of the detergentcomposition. The particular enzyme chosen for use in the process andproducts of this invention depends upon the conditions of final utility,including the physical product form, use pH, use temperature, and soiltypes to be degraded or altered. The enzyme can be chosen to provideoptimum activity and stability for any given set of utility conditions.In one aspect, the xylanases of the present invention are active in thepH ranges of from about 4 to about 12 and in the temperature range offrom about 20° C. to about 95° C. The detergents of the invention cancomprise cationic, semi-polar nonionic or zwitterionic surfactants; or,mixtures thereof.

Xylanases of the invention can be formulated into powdered and liquiddetergents, disinfectants or cleansers having pH between 4.0 and 12.0 atlevels of about 0.01 to about 5% (preferably 0.1% to 0.5%) by weight.These detergent, disinfectant or cleanser compositions can also includeother enzymes such as xylanases, cellulases, lipases, esterases,proteases, or endoglycosidases, endo-beta.-1,4-glucanases,beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases,catalases, laccases, amylases, glucoamylases, pectinases, reductases,oxidases, phenoloxidases, ligninases, pullulanases, arabinanases,hemicellulases, mannanases, xyloglucanases, xylanases, pectin acetylesterases, rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases. Thesedetergent, disinfectant or cleanser compositions can also include dyes,colorants, odorants, bleaches, buffers, builders, enzyme “enhancingagents” (see, e.g., U.S. patent application no. 20030096394) andstabilizers.

The addition of xylanases of the invention to conventional cleaningcompositions does not create any special use limitation. In other words,any temperature and pH suitable for the detergent is also suitable forthe compositions of the invention as long as the enzyme is active at ortolerant of the pH and/or temperature of the intended use. In addition,the xylanases of the invention can be used in a cleaning compositionwithout detergents, again either alone or in combination with buildersand stabilizers.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions.

In one aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. A xylanase of the invention maybe included as a detergent, disinfectant or cleanser additive. Thedetergent, disinfectant or cleanser composition of the invention may,for example, be formulated as a hand or machine laundry detergent,disinfectant or cleanser composition comprising a polypeptide of theinvention. A laundry additive suitable for pretreatment of stainedfabrics can comprise a polypeptide of the invention. A fabric softenercomposition can comprise a xylanase of the invention. Alternatively, axylanase of the invention can be formulated as a detergent, disinfectantor cleanser composition for use in general household hard surfacecleaning operations. In alternative aspects, detergent, disinfectant orcleanser additives and detergent, disinfectant or cleanser compositionsof the invention may comprise one or more other enzymes such as axylanase, a lipase, a protease, a cutinase, an esterase, anotherxylanase, a carbohydrase, a cellulase, a pectinase, a mannanase, anarabinase, a galactanase, a xylanase, an oxidase, e.g., a lactase,and/or a peroxidase (see also, above). The properties of the enzyme(s)of the invention are chosen to be compatible with the selected detergent(i.e. pH-optimum, compatibility with other enzymatic and non-enzymaticingredients, etc.) and the enzyme(s) is present in effective amounts. Inone aspect, xylanase enzymes of the invention are used to removemalodorous materials from fabrics. Various detergent compositions andmethods for making them that can be used in practicing the invention aredescribed in, e.g., U.S. Pat. Nos. 6,333,301; 6,329,333; 6,326,341;6,297,038; 6,309,871; 6,204,232; 6,197,070; 5,856,164.

When formulated as compositions suitable for use in a laundry machinewashing method, the xylanases of the invention can comprise both asurfactant and a builder compound. They can additionally comprise one ormore detergent components, e.g., organic polymeric compounds, bleachingagents, additional enzymes, suds suppressors, dispersants, lime-soapdispersants, soil suspension and anti-redeposition agents and corrosioninhibitors. Laundry compositions of the invention can also containsoftening agents, as additional detergent components. Such compositionscontaining carbohydrase can provide fabric cleaning, stain removal,whiteness maintenance, softening, color appearance, dye transferinhibition and sanitization when formulated as laundry detergentcompositions.

The density of the laundry detergent, disinfectant or cleansercompositions of the invention can range from about 200 to 1500 g/liter,or, about 400 to 1200 g/liter, or, about 500 to 950 g/liter, or, 600 to800 g/liter, of composition; this can be measured at about 20° C.

In one aspect, the “compact” form of laundry detergent, disinfectant orcleanser compositions of the invention is best reflected by density and,in terms of composition, by the amount of inorganic filler salt.Inorganic filler salts are conventional ingredients of detergentcompositions in powder form. In conventional detergent compositions, thefiller salts are present in substantial amounts, typically 17% to 35% byweight of the total composition. In one aspect of the compactcompositions, the filler salt is present in amounts not exceeding 15% ofthe total composition, or, not exceeding 10%, or, not exceeding 5% byweight of the composition. The inorganic filler salts can be selectedfrom the alkali and alkaline-earth-metal salts of sulphates andchlorides, e.g., sodium sulphate.

Liquid detergent compositions of the invention can also be in a“concentrated form.” In one aspect, the liquid detergent, disinfectantor cleanser compositions can contain a lower amount of water, comparedto conventional liquid detergents, disinfectants or cleansers. Inalternative aspects, the water content of the concentrated liquiddetergent is less than 40%, or, less than 30%, or, less than 20% byweight of the detergent, disinfectant or cleanser composition.Detergent, disinfectant or cleanser compounds of the invention cancomprise formulations as described in WO 97/01629.

Xylanases of the invention can be useful in formulating variousdetergent, cleaning, disinfectant or cleanser compositions. A number ofknown compounds are suitable surfactants including nonionic, anionic,cationic, or zwitterionic detergents, can be used, e.g., as disclosed inU.S. Pat. Nos. 4,404,128; 4,261,868; 5,204,015. In addition, xylanasescan be used, for example, in bar or liquid soap applications, dish careformulations, contact lens cleaning solutions or products, peptidehydrolysis, waste treatment, textile applications, as fusion-cleavageenzymes in protein production, and the like. Xylanases may provideenhanced performance in a detergent composition as compared to anotherdetergent xylanase, that is, the enzyme group may increase cleaning ofcertain enzyme sensitive stains such as grass or blood, as determined byusual evaluation after a standard wash cycle. Xylanases can beformulated into known powdered and liquid detergents having pH between6.5 and 12.0 at levels of about 0.01 to about 5% (for example, about0.1% to 0.5%) by weight. These detergent cleaning compositions can alsoinclude other enzymes such as known xylanases, xylanases, proteases,amylases, cellulases, mannanases, lipases or endoglycosidases, redoxenzymes such as catalases and laccases, as well as builders,stabilizers, fragrances and pigments.

In one aspect, the invention provides detergent, disinfectant orcleanser compositions having xylanase activity (a xylanase of theinvention) for use with fruit, vegetables and/or mud and clay compounds(see, for example, U.S. Pat. No. 5,786,316).

In another aspect of the invention, the xylanases of the invention canalso be used in any detergent, disinfectant or cleanser (cleaningsolution) manufacturing process, wherein the xylanase is used as anantimicrobial or microbial repellent. Alternatively, the xylanases ofthe invention can be used in any cleasing or washing process, whereinthe xylanase is used as an antimicrobial or microbial repellent. Inanother aspect of the invention, the xylanase of the invention can beincluded in any detergent or cleanser composition, wherein the xylanasesof the invention act as an antimicrobial or microbial repellent in thecomposition.

Treating Foods and Food Processing

The xylanases of the invention have numerous applications in foodprocessing industry. For example, in one aspect, the xylanases of theinvention are used to improve the extraction of oil from oil-rich plantmaterial, e.g., oil-rich seeds, for example, soybean oil from soybeans,olive oil from olives, rapeseed oil from rapeseed and/or sunflower oilfrom sunflower seeds.

The xylanases of the invention can be used for separation of componentsof plant cell materials. For example, xylanases of the invention can beused in the separation of xylan-rich material (e.g., plant cells) intocomponents. In one aspect, xylanases of the invention can be used toseparate xylan-rich or oil-rich crops into valuable protein and oil andhull fractions. The separation process may be performed by use ofmethods known in the art.

The xylanases of the invention can be used in the preparation of fruitor vegetable juices, syrups, extracts and the like to increase yield.The xylanases of the invention can be used in the enzymatic treatment(e.g., hydrolysis of xylan-comprising plant materials) of various plantcell wall-derived materials or waste materials, e.g. from cereals,grains, wine or juice production, or agricultural residues such asvegetable hulls, bean hulls, sugar beet pulp, olive pulp, potato pulp,and the like. The xylanases of the invention can be used to modify theconsistency and appearance of processed fruit or vegetables. Thexylanases of the invention can be used to treat plant material tofacilitate processing of plant material, including foods, facilitatepurification or extraction of plant components. The xylanases of theinvention can be used to improve feed value, decrease the water bindingcapacity, improve the degradability in waste water plants and/or improvethe conversion of plant material to ensilage, and the like.

In one aspect, xylanases of the invention are used in bakingapplications, e.g., cookies and crackers, to hydrolyze xylans such asarabinoxylans. In one aspect, xylanases of the invention are used tocreate non-sticky doughs that are not difficult to machine and to reducebiscuit size. Xylanases of the invention can be used to hydrolyzearabinoxylans to prevent rapid rehydration of the baked productresulting in loss of crispiness and reduced shelf-life. In one aspect,xylanases of the invention are used as additives in dough processing. Inone aspect, xylanases of the invention are used in dough conditioning,wherein in one aspect the xylanases possess high activity over atemperature range of about 25-35° C. and at near neutral pH (7.0-7.5).In one aspect, dough conditioning enzymes can be inactivated at theextreme temperatures of baking (>500° F.). The enzymes of the inventioncan be used in conjunction with any dough processing protocol, e.g., asin U.S. Patent App. No. 20050281916.

In one aspect, xylanases of the invention are used as additives in doughprocessing to perform optimally under dough pH and temperatureconditions. In one aspect, an enzyme of the invention is used for doughconditioning. In one aspect, a xylanase of the invention possesses highactivity over a temperature range of 25-35° C. and at near neutral pH(7.0-7.5). In one aspect, the enzyme is inactivated at the extremetemperatures of baking, for example, >500° F.

In another aspect of the invention, the xylanases of the invention canalso be used in any food or beverage treatment or food or beverageprocessing process, wherein the xylanase is used as an antimicrobial ormicrobial repellent. In another aspect of the invention, the xylanase ofthe invention can be included in any food or bevereage composition,wherein the xylanases of the invention act as an antimicrobial ormicrobial repellent in the composition.

Animal Feeds and Food or Feed or Food Additives (Supplements)

The invention provides methods for treating animal feeds and foods andfood or feed additives (supplements) using xylanases of the invention,animals including mammals (e.g., humans), birds, fish and the like. Theinvention provides animal feeds, foods, and additives (supplements)comprising xylanases of the invention. In one aspect, treating animalfeeds, foods and additives using xylanases of the invention can help inthe availability of nutrients, e.g., starch, protein, and the like, inthe animal feed or additive (supplements). By breaking down difficult todigest proteins or indirectly or directly unmasking starch (or othernutrients), the xylanase makes nutrients more accessible to otherendogenous or exogenous enzymes. The xylanase can also simply cause therelease of readily digestible and easily absorbed nutrients and sugars.

When added to animal feed, xylanases of the invention improve the invivo break-down of plant cell wall material partly due to a reduction ofthe intestinal viscosity (see, e.g., Bedford et al., Proceedings of the1st Symposium on Enzymes in Animal Nutrition, 1993, pp. 73-77), wherebya better utilization of the plant nutrients by the animal is achieved.Thus, by using xylanases of the invention in feeds the growth rateand/or feed conversion ratio (i.e. the weight of ingested feed relativeto weight gain) of the animal is improved.

The animal feed additive of the invention may be a granulated enzymeproduct which may readily be-mixed with feed components. Alternatively,feed additives of the invention can form a component of a pre-mix. Thegranulated enzyme product of the invention may be coated or uncoated.The particle size of the enzyme granulates can be compatible with thatof feed and pre-mix components. This provides a safe and convenient meanof incorporating enzymes into feeds. Alternatively, the animal feedadditive of the invention may be a stabilized liquid composition. Thismay be an aqueous or oil-based slurry. See, e.g., U.S. Pat. No.6,245,546.

Xylanases of the present invention, in the modification of animal feedor a food, can process the food or feed either in vitro (by modifyingcomponents of the feed or food) or in vivo. Xylanases can be added toanimal feed or food compositions containing high amounts of xylans, e.g.feed or food containing plant material from cereals, grains and thelike. When added to the feed or food the xylanase significantly improvesthe in vivo break-down of xylan-containing material, e.g., plant cellwalls, whereby a better utilization of the plant nutrients by the animal(e.g., human) is achieved. In one aspect, the growth rate and/or feedconversion ratio (i.e. the weight of ingested feed relative to weightgain) of the animal is improved. For example a partially or indigestiblexylan-comprising protein is fully or partially degraded by a xylanase ofthe invention, e.g. in combination with another enzyme, e.g.,beta-galactosidase, to peptides and galactose and/or galactooligomers.These enzyme digestion products are more digestible by the animal. Thus,xylanases of the invention can contribute to the available energy of thefeed or food. Also, by contributing to the degradation ofxylan-comprising proteins, a xylanase of the invention can improve thedigestibility and uptake of carbohydrate and non-carbohydrate feed orfood constituents such as protein, fat and minerals.

In another aspect, xylanase of the invention can be supplied byexpressing the enzymes directly in transgenic feed crops (as, e.g.,transgenic plants, seeds and the like), such as grains, cereals, corn,soy bean, rape seed, lupin and the like. As discussed above, theinvention provides transgenic plants, plant parts and plant cellscomprising a nucleic acid sequence encoding a polypeptide of theinvention. In one aspect, the nucleic acid is expressed such that thexylanase of the invention is produced in recoverable quantities. Thexylanase can be recovered from any plant or plant part. Alternatively,the plant or plant part containing the recombinant polypeptide can beused as such for improving the quality of a food or feed, e.g.,improving nutritional value, palatability, and rheological properties,or to destroy an antinutritive factor.

In one aspect, the invention provides methods for removingoligosaccharides from feed prior to consumption by an animal subjectusing a xylanase of the invention. In this process a feed is formedhaving an increased metabolizable energy value. In addition to xylanasesof the invention, galactosidases, cellulases and combinations thereofcan be used. In one aspect, the enzyme is added in an amount equal tobetween about 0.1% and 1% by weight of the feed material. In one aspect,the feed is a cereal, a wheat, a grain, a soybean (e.g., a groundsoybean) material. See, e.g., U.S. Pat. No. 6,399,123.

In another aspect, the invention provides methods for utilizing xylanaseas a nutritional supplement in the diets of animals by preparing anutritional supplement containing a recombinant xylanase enzymecomprising at least thirty contiguous amino acids of a sequence of theinvention, and administering the nutritional supplement to an animal toincrease the utilization of xylan contained in food ingested by theanimal.

In yet another aspect, the invention provides an edible pelletizedenzyme delivery matrix and method of use for delivery of xylanase to ananimal, for example as a nutritional supplement. The enzyme deliverymatrix readily releases a xylanase enzyme, such as one having an aminoacid sequence of the invention, or at least 30 contiguous amino acidsthereof, in aqueous media, such as, for example, the digestive fluid ofan animal. The invention enzyme delivery matrix is prepared from agranulate edible carrier selected from such components as grain germthat is spent of oil, hay, alfalfa, timothy, soy hull, sunflower seedmeal, corn meal, soy meal, wheat midd, and the like, that readilydisperse the recombinant enzyme contained therein into aqueous media. Inuse, the edible pelletized enzyme delivery matrix is administered to ananimal to delivery of xylanase to the animal. Suitable grain-basedsubstrates may comprise or be derived from any suitable edible grain,such as wheat, corn, soy, sorghum, alfalfa, barley, and the like. Anexemplary grain-based substrate is a corn-based substrate. The substratemay be derived from any suitable part of the grain, but is preferably agrain germ approved for animal feed use, such as corn germ that isobtained in a wet or dry milling process. The grain germ preferablycomprises spent germ, which is grain germ from which oil has beenexpelled, such as by pressing or hexane or other solvent extraction.Alternatively, the grain germ is expeller extracted, that is, the oilhas been removed by pressing.

The enzyme delivery matrix of the invention is in the form of discreteplural particles, pellets or granules. By “granules” is meant particlesthat are compressed or compacted, such as by a pelletizing, extrusion,or similar compacting to remove water from the matrix. Such compressionor compacting of the particles also promotes intraparticle cohesion ofthe particles. For example, the granules can be prepared by pelletizingthe grain-based substrate in a pellet mill. The pellets prepared therebyare ground or crumbled to a granule size suitable for use as an adjuvantin animal feed. Since the matrix is itself approved for use in animalfeed, it can be used as a diluent for delivery of enzymes in animalfeed.

The enzyme delivery matrix can be in the form of granules having agranule size ranging from about 4 to about 400 mesh (USS); morepreferably, about 8 to about 80 mesh; and most preferably about 14 toabout 20 mesh. If the grain germ is spent via solvent extraction, use ofa lubricity agent such as corn oil may be necessary in the pelletizer,but such a lubricity agent ordinarily is not necessary if the germ isexpeller extracted. In other aspects of the invention, the matrix isprepared by other compacting or compressing processes such as, forexample, by extrusion of the grain-based substrate through a die andgrinding of the extrudate to a suitable granule size.

The enzyme delivery matrix may further include a polysaccharidecomponent as a cohesiveness agent to enhance the cohesiveness of thematrix granules. The cohesiveness agent is believed to provideadditional hydroxyl groups, which enhance the bonding between grainproteins within the matrix granule. It is further believed that theadditional hydroxyl groups so function by enhancing the hydrogen bondingof proteins to starch and to other proteins. The cohesiveness agent maybe present in any amount suitable to enhance the cohesiveness of thegranules of the enzyme delivery matrix. Suitable cohesiveness agentsinclude one or more of dextrins, maltodextrins, starches, such as cornstarch, flours, cellulosics, hemicellulosics, and the like. For example,the percentage of grain germ and cohesiveness agent in the matrix (notincluding the enzyme) is 78% corn germ meal and 20% by weight of cornstarch.

Because the enzyme-releasing matrix of the invention is made frombiodegradable materials and contains moisture, the matrix may be subjectto spoilage, such as by molding. To prevent or inhibit such molding, thematrix may include a mold inhibitor, such as a propionate salt, whichmay be present in any amount sufficient to inhibit the molding of theenzyme-releasing matrix, thus providing a delivery matrix in a stableformulation that does not require refrigeration.

The xylanase enzyme contained in the invention enzyme delivery matrixand methods is preferably a thermostable xylanase, as described herein,so as to resist inactivation of the xylanase during manufacture whereelevated temperatures and/or steam may be employed to prepare thepelletized enzyme delivery matrix. During digestion of feed containingthe invention enzyme delivery matrix, aqueous digestive fluids willcause release of the active enzyme. Other types of thermostable enzymesand nutritional supplements that are thermostable can also beincorporated in the delivery matrix for release under any type ofaqueous conditions.

A coating can be applied to the invention enzyme matrix particles formany different purposes, such as to add a flavor or nutrition supplementto animal feed, to delay release of animal feed supplements and enzymesin gastric conditions, and the like. Or, the coating may be applied toachieve a functional goal, for example, whenever it is desirable to slowrelease of the enzyme from the matrix particles or to control theconditions under which the enzyme will be released. The composition ofthe coating material can be such that it is selectively broken down byan agent to which it is susceptible (such as heat, acid or base, enzymesor other chemicals). Alternatively, two or more coatings susceptible todifferent such breakdown agents may be consecutively applied to thematrix particles.

The invention is also directed towards a process for preparing anenzyme-releasing matrix. In accordance with the invention, the processcomprises providing discrete plural particles of a grain-based substratein a particle size suitable for use as an enzyme-releasing matrix,wherein the particles comprise a xylanase enzyme encoded by an aminoacid sequence of the invention or at least 30 consecutive amino acidsthereof. Preferably, the process includes compacting or compressing theparticles of enzyme-releasing matrix into granules, which can beaccomplished by pelletizing. The mold inhibitor and cohesiveness agent,when used, can be added at any suitable time, and can be mixed with thegrain-based substrate in the desired proportions prior to pelletizing ofthe grain-based substrate. Moisture content in the pellet mill feed canbe in the ranges set forth above with respect to the moisture content inthe finished product, and can be about 14-15%. In one aspect, moistureis added to the feedstock in the form of an aqueous preparation of theenzyme to bring the feedstock to this moisture content. The temperaturein the pellet mill can be brought to about 82° C. with steam. The pelletmill may be operated under any conditions that impart sufficient work tothe feedstock to provide pellets. The pelleting process itself is acost-effective process for removing water from the enzyme-containingcomposition.

In one aspect, the pellet mill is operated with a 1/8 in. by 2 inch dieat 100 lb./min. pressure at 82° C. to provide pellets, which then arecrumbled in a pellet mill crumbler to provide discrete plural particleshaving a particle size capable of passing through an 8 mesh screen butbeing retained on a 20 mesh screen.

The thermostable xylanases of the invention can be used in the pelletsof the invention. They can have high optimum temperatures and high heatresistance such that an enzyme reaction at a temperature not hithertocarried out can be achieved. The gene encoding the xylanase according tothe present invention (e.g. as set forth in any of the sequences in theinvention) can be used in preparation of xylanases (e.g. using GSSM asdescribed herein) having characteristics different from those of thexylanases of the invention (in terms of optimum pH, optimum temperature,heat resistance, stability to solvents, specific activity, affinity tosubstrate, secretion ability, translation rate, transcription controland the like). Furthermore, a polynucleotide of the invention may beemployed for screening of variant xylanases prepared by the methodsdescribed herein to determine those having a desired activity, such asimproved or modified thermostability or thermotolerance. For example,U.S. Pat. No. 5,830,732, describes a screening assay for determiningthermotolerance of a xylanase.

In another aspect of the invention, the xylanases of the invention canalso be used in any animal feed, animal food or feed additive productionprocess, wherein the xylanase is used as an antimicrobial or microbialrepellent. In another aspect of the invention, the xylanase of theinvention can be included in any animal feed, animal food or feedadditive composition, wherein the xylanases of the invention act as anantimicrobial or microbial repellent in the composition.

Waste Treatment

The xylanases of the invention can be used in a variety of otherindustrial applications, e.g., in waste treatment. For example, in oneaspect, the invention provides a solid waste digestion process usingxylanases of the invention. The methods can comprise reducing the massand volume of substantially untreated solid waste. Solid waste can betreated with an enzymatic digestive process in the presence of anenzymatic solution (including xylanases of the invention) at acontrolled temperature. This results in a reaction without appreciablebacterial fermentation from added microorganisms. The solid waste isconverted into a liquefied waste and any residual solid waste. Theresulting liquefied waste can be separated from said any residualsolidified waste. See e.g., U.S. Pat. No. 5,709,796.

In another aspect of the invention, the xylanases of the invention canalso be used in any waste treatment process, wherein the xylanase isused as an antimicrobial or microbial repellent. In another aspect ofthe invention, the xylanase of the invention can be included in anywaste treatment composition, wherein the xylanases of the invention actas an antimicrobial or microbial repellent in the composition.

Oral Care Products

The invention provides oral care product comprising xylanases of theinvention, including the enzyme mixtures or “cocktails” of theinvention. Exemplary oral care products include toothpastes, dentalcreams, gels or tooth powders, odontics, mouth washes, pre- or postbrushing rinse formulations, chewing gums, lozenges, or candy. See,e.g., U.S. Pat. No. 6,264,925.

In another aspect of the invention, the xylanases of the invention,including the enzyme mixtures or “cocktails” of the invention, can alsobe used in any oral care manufacturing process, wherein the xylanase isused as an antimicrobial or microbial repellent. In another aspect ofthe invention, the xylanase of the invention, including the enzymemixtures or “cocktails” of the invention, can be included in any oralcare composition, wherein the xylanases of the invention act as anantimicrobial or microbial repellent in the composition.

Brewing and Fermenting

The invention provides methods of brewing (e.g., fermenting) beercomprising xylanases of the invention, including the enzyme mixtures or“cocktails” of the invention. In one exemplary process,starch-containing raw materials are disintegrated and processed to forma malt. A xylanase of the invention is used at any point in thefermentation process. For example, xylanases of the invention can beused in the processing of barley malt. The major raw material of beerbrewing is barley malt. This can be a three stage process. First, thebarley grain can be steeped to increase water content, e.g., to aroundabout 40%. Second, the grain can be germinated by incubation at 15 to25° C. for 3 to 6 days when enzyme synthesis is stimulated under thecontrol of gibberellins. In one aspect, xylanases of the invention areadded at this (or any other) stage of the process. Xylanases of theinvention can be used in any beer or alcoholic beverage producingprocess, as described, e.g., in U.S. Pat. Nos. 5,762,991; 5,536,650;5,405,624; 5,021,246; 4,788,066.

In one aspect, an enzyme of the invention is used to improvefilterability and wort viscosity and to obtain a more completehydrolysis of endosperm components. Use of an enzyme of the inventionwould also increase extract yield. The process of brewing involvesgermination of the barley grain (malting) followed by the extraction andthe breakdown of the stored carbohydrates to yield simple sugars thatare used by yeast for alcoholic fermentation. Efficient breakdown of thecarbohydrate reserves present in the barley endosperm and brewingadjuncts requires the activity of several different enzymes.

In one aspect, an enzyme of the invention has activity in slightlyacidic pH (e.g., 5.5-6.0) in, e.g., the 40° C. to 70° C. temperaturerange; and, in one aspect, with inactivation at 95° C. Activity undersuch conditions would be optimal, but are not an essential requirementfor efficacy. In one aspect, an enzyme of the invention has activitybetween 40-75° C., and pH 5.5-6.0; stable at 70° for at least 50minutes, and, in one aspect, is inactivated at 96-100° C. Enzymes of theinvention can be used with other enzymes, e.g., beta-1,4-endoglucanasesand amylases.

In another aspect of the invention, the xylanases of the invention,including the enzyme mixtures or “cocktails” of the invention, can alsobe used in any brewing or fermentation process, wherein the xylanase isused as an antimicrobial or microbial repellent. In another aspect ofthe invention, the xylanase of the invention can be included in anybrewed or fermented composition, wherein the xylanases of the inventionact as an antimicrobial or microbial repellent in the composition.

Biomass Conversion and Biofuel Production

The invention provides methods and processes for biomass conversion,e.g., to a biofuel, such as bioethanol, biomethanol, biopropanol and/orbiobutanol and the like, using enzymes of the invention, including theenzyme mixtures or “cocktails” of the invention. Thus, the inventionprovides fuels, e.g., biofuels, such as bioethanols, comprising apolypeptide of the invention, including the enzyme mixtures or“cocktails” of the invention, or a polypeptide encoded by a nucleic acidof the invention. In alternative aspects, the fuel is derived from aplant material, which optionally comprises potatoes, soybean (rapeseed),barley, rye, corn, oats, wheat, beets or sugar cane, and optionally thefuel comprises a bioethanol or a gasoline-ethanol mix.

The invention provides methods for making a fuel comprising contacting acomposition comprising a xylan, hemicellulose, cellulose or afermentable sugar with a polypeptide of the invention, or a polypeptideencoded by a nucleic acid of the invention, or any one of the mixturesor “cocktails” or products of manufacture of the invention. Inalternative embodiments, the composition comprising a xylan,hemicellulose, a cellulose or a fermentable sugar comprises a plant,plant product or plant derivative, and the plant or plant product cancomprise cane sugar plants or plant products, beets or sugarbeets,wheat, corn, soybeans, potato, rice or barley. In alternativeembodiments, the polypeptide has activity comprising catalyzinghydrolysis of internal β-1,4-xylosidic linkages or endo-β-1,4-glucanaselinkages; and/or degrading a linear polysaccharide beta-1,4-xylan intoxylose. In one aspect, the fuel comprises a bioethanol or agasoline-ethanol mix, or a biopropanol or a gasoline-propanol mix, or abiobutanol or a gasoline-butanol mix, or a biomethanol or agasoline-methanol mix, or any combination thereof.

The invention provides methods for making bioethanol, biobutanol,biomethanol and/or a biopropanol comprising contacting a compositioncomprising a xylan, hemi-cellulose, cellulose or a fermentable sugarwith a polypeptide of the invention, or a polypeptide encoded by anucleic acid of the invention, or any one of the mixtures or “cocktails”or products of manufacture of the invention. In alternative embodiments,the composition comprising a cellulose or a fermentable sugar comprisesa plant, plant product or plant derivative, and the plant or plantproduct can comprise cane sugar plants or plant products, beets orsugarbeets, wheat, corn, soybeans, potato, rice or barley, and thepolypeptide can have activity comprising cellulase, glucanase,cellobiohydrolase, beta-glucosidase, xylanase, mannanse, β-xylosidase,and/or arabinofuranosidase activity.

The invention provides enzyme ensembles, or “cocktails”, fordepolymerization of cellulosic and hemicellulosic polymers, xylans, andpolysaccharides to metabolizeable carbon moieties comprising apolypeptide of the invention, or a polypeptide encoded by a nucleic acidof the invention. In alternative embodiments, the polypeptide hasactivity comprising catalyzing hydrolysis of internal β-1,4-xylosidiclinkages or endo-β-1,4-glucanase linkages; and/or degrading a linearpolysaccharide beta-1,4-xylan into xylose. The enzyme ensembles, or“cocktails”, of the invention can be in the form of a composition (e.g.,a formulation, liquid or solid), e.g., as a product of manufacture. Theinvention further enzymes, enzyme ensembles, or “cocktails” fordepolymerization of cellulosic and hemicellulosic polymers, xylans andpolysaccharides, to simpler moieties, such as sugars, which are thenmicrobially fermented to generate products such as succinic acid, lacticacid, or acetic acid.

The invention provides compositions (including products of manufacture,enzyme ensembles, or “cocktails”) comprising a mixture (or “cocktail”)of hemicellulose- and cellulose-hydrolyzing enzymes, wherein thexylan-hydrolyzing enzymes comprise at least one of each of a xylanase ofthe invention and at least one, several or all of a cellulase,glucanase, a cellobiohydrolase and/or a β-glucosidase. In alternativeembodiments, the xylan-hydrolyzing and/or hemicellulose-hydrolyzingmixtures of the invention comprise at least one of each of a xylanase ofthe invention and at least one or both of a β-xylosidase and/or anarabinofuranosidase.

The invention provides compositions (including products of manufacture,enzyme ensembles, or “cocktails”) comprising a mixture (or “cocktail”)of xylan-hydrolyzing, hemicellulose- and/or cellulose-hydrolyzingenzymes comprising at least one, several or all of a cellulase, aglucanase, a cellobiohydrolase and/or an arabinofuranosidase, and axylanase of this invention.

The invention provides compositions (including products of manufacture,enzyme ensembles, or “cocktails”) comprising mixture (or “cocktail”) ofxylan-hydrolyzing, hemicellulose- and/or cellulose-hydrolyzing enzymescomprising at least one, several or all of a cellulase, a glucanase; acellobiohydrolase; an arabinofuranosidase; a xylanase; a β-glucosidase;a β-xylosidase; and at least one enzyme of the invention.

The invention provides compositions (including products of manufacture,enzyme ensembles, or “cocktails”) comprising mixture (or “cocktail”) ofenzymes comprising, in addition to at least one enzyme of the invention:(1) a glucanase which cleaves internal β-1,4 linkages resulting inshorter glucooligosaccharides, (2) a cellobiohydrolase which acts in an“exo” manner processively releasing cellobiose units (β-1,4glucose-glucose disaccharide), and/or (3) a β-glucosidase for releasingglucose monomer from short cellooligosaccharides (e.g. cellobiose).

Biomass Conversion and Production of Clean Bio Fuels

The invention provides compositions and processes using enzymes of thisinvention, including mixtures, or “cocktails” of enzymes of theinvention, for the conversion of a biomass, or any organic material,e.g., any xylan-comprising or lignocellulosic material (e.g., anycomposition comprising a xylan, cellulose, hemicellulose and/or lignin),to a fuel, such as a biofuel (e.g., bioethanol, biobutanol, biomethanoland/or a biopropanol), including biodiesels, in addition to feeds,foods, food or feed supplements (additives), pharmaceuticals andchemicals. Thus, the compositions and methods of the invention provideeffective and sustainable alternatives or adjuncts to use ofpetroleum-based products, e.g., as a mixture of a biofuel (e.g.,bioethanol, biobutanol, biomethanol and/or a biopropanol) and gasolineand/or diesel fuel.

The invention provides cells and/or organisms expressing enzymes of theinvention (e.g., wherein the cells or organisms comprise as heterologousnucleic acids a sequence of this invention) for participation inchemical cycles involving natural biomass (e.g., plant) conversion. Inone aspect, enzymes and methods for the conversion are used in enzymeensembles (or “cocktails”) for the efficient depolymerization ofxylan-comprising compositions, or xylan, cellulosic and hemicellulosicpolymers, to metabolizeable carbon moieties. The invention providesmethods for discovering and implementing the most effective of enzymesto enable these important new “biomass conversion” and alternativeenergy industrial processes.

The invention provides methods, enzymes and mixtures of enzymes or“cocktails” of the invention, for processing a material, e.g. a biomassmaterial, comprising a cellooligsaccharide, an arabinoxylan oligomer, alignin, a lignocellulose, a xylan, a glucan, a cellulose and/or afermentable sugar comprising contacting the composition with apolypeptide of the invention, or a polypeptide encoded by a nucleic acidof the invention, wherein optionally the material is derived from anagricultural crop (e.g., wheat, barley, potatoes, switchgrass, poplarwood), is a byproduct of a food or a feed production, is alignocellulosic waste product, or is a plant residue or a waste paper orwaste paper product, and optionally the plant residue comprise stems,leaves, hulls, husks, corn or corn cobs, corn stover, corn fiber, hay,straw (e.g. rice straw or wheat straw), sugarcane bagasse, sugar beetpulp, citrus pulp, and citrus peels, wood, wood thinnings, wood chips,wood pulp, pulp waste, wood waste, wood shavings and sawdust,construction and/or demolition wastes and debris (e.g. wood, woodshavings and sawdust), and optionally the paper waste comprisesdiscarded or used photocopy paper, computer printer paper, notebookpaper, notepad paper, typewriter paper, newspapers, magazines, cardboardand paper-based packaging materials, and recycled paper materials. Inaddition, urban wastes, e.g. the paper fraction of municipal solidwaste, municipal wood waste, and municipal green waste, along with othermaterials containing sugar, starch, and/or cellulose can be used.Optionally the processing of the material, e.g. the biomass material,generates a bioalcohol, e.g., a bioethanol, biomethanol, biobutanol orbiopropanol.

Alternatively, the polypeptide of the invention may be expressed in thebiomass plant material or feedstock itself.

The methods of the invention also include taking the converted biomass(e.g., lignocellulosic) material (processed by enzymes of the invention)and making it into a fuel (e.g. a biofuel such as a bioethanol,biobutanol, biomethanol, a biopropanol, or a biodiesel) by fermentationand/or by chemical synthesis. In one aspect, the produced sugars arefermented and/or the non-fermentable products are gasified.

The methods of the invention also include converting algae, virginvegetable oils, waste vegetable oils, animal fats and greases (e.g.tallow, lard, and yellow grease), or sewage, using enzymes of theinvention, and making it into a fuel (e.g. a bioalcohol, e.g., abioethanol, biomethanol, biobutanol or biopropanol, or biodiesel) byfermentation and/or by chemical synthesis or conversion.

The enzymes of the invention (including, for example, organisms, such asmicroorganisms, e.g., fungi, yeast or bacteria, and plants and plantcells and plant parts, e.g., seeds, making and in some aspects secretingrecombinant enzymes of the invention) can be used in orincluded/integrated at any stage of any organic matter/biomassconversion process, e.g., at any one step, several steps, or included inall of the steps, or all of the following methods of biomass conversionprocesses, or all of these biofuel alternatives:

Direct combustion: the burning of material by direct heat and is thesimplest biomass technology; can be very economical if a biomass sourceis nearby.

-   -   1 Pyrolysis: is the thermal degradation of biomass by heat in        the absence of oxygen. In one aspect, biomass is heated to a        temperature between about 800 and 1400 degrees Fahrenheit, but        no oxygen is introduced to support combustion resulting in the        creation of gas, fuel oil and charcoal.    -   2 Gasification: biomass can be used to produce methane through        heating or anaerobic digestion. Syngas, a mixture of carbon        monoxide and hydrogen, can be derived from biomass.

Landfill Gas: is generated by the decay (anaerobic digestion) of buriedgarbage in landfills. When the organic waste decomposes, it generatesgas consisting of approximately 50% methane, the major component ofnatural gas.

Anaerobic digestion: converts organic matter to a mixture of methane,the major component of natural gas, and carbon dioxide. In one aspect,biomass such as waterwaste (sewage), manure, or food processing waste,is mixed with water and fed into a digester tank without air.

Fermentation

-   -   Alcohol Fermentation: fuel alcohol is produced by converting        cellulosic mass and/or starch to sugar, fermenting the sugar to        alcohol, then separating the alcohol water mixture by        distillation. Feedstocks such as dedicated crops (e.g., wheat,        barley, potatoes, switchgrass, poplar wood), agricultural        residues and wastes (e.g. rice straw, corn stover, wheat straw,        sugarcane bagasse, rice hulls, corn fiber, sugar beet pulp,        citrus pulp, and citrus peels), forestry wastes (e.g. hardwood        and softwood thinnings, hardwood and softwood residues from        timber operations, wood shavings, and sawdust), urban wastes        (e.g. paper fraction of municipal solid waste, municipal wood        waste, municipal green waste), wood wastes (e.g. saw mill waste,        pulp mill waste, construction waste, demolition waste, wood        shavings, and sawdust), and waste paper or other materials        containing sugar, starch, and/or cellulose can be converted to        sugars and then to alcohol by fermentation with yeast.        Alternatively, materials containing sugars can be converted        directly to alcohol by fermentation.

Transesterification: An exemplary reaction for converting oil tobiodiesel is called transesterification. The transesterification processreacts an alcohol (like methanol) with the triglyceride oils containedin vegetable oils, animal fats, or recycled greases, forming fatty acidalkyl esters (biodiesel) and glycerin. The reaction requires heat and astrong base catalyst, such as sodium hydroxide or potassium hydroxide.

Biodiesel: Biodiesel is a mixture of fatty acid alkyl esters made fromvegetable oils, animal fats or recycled greases. Biodiesel can be usedas a fuel for vehicles in its pure form, but it is usually used as apetroleum diesel additive to reduce levels of particulates, carbonmonoxide, hydrocarbons and air toxics from diesel-powered vehicles.

Hydrolysis: includes hydrolysis of a compound, e.g., a biomass, such asa lignocellulosic material, catalyzed using an enzyme of the instantinvention.

Cogeneration: is the simultaneous production of more than one form ofenergy using a single fuel and facility. In one aspect, biomasscogeneration has more potential growth than biomass generation alonebecause cogeneration produces both heat and electricity.

In one aspect, the polypeptides of the invention have sufficientenzymatic activity, e.g. a xylanase, a mannanase and/or a glucanaseactivity, for, or can be used with other enzymes in a process for,generating a biodiesel or a fuel, (e.g. a bioalcohol, e.g., abioethanol, biomethanol, biobutanol or biopropanol, or biodiesel) froman organic material, e.g., a biomass, such as compositions derived fromplants and animals, including any agricultural crop or other renewablefeedstock, an agricultural residue or an animal waste, the organiccomponents of municipal and industrial wastes, or construction ordemolition wastes or debris, or microorganisms such as algae or yeast.

In one aspect, polypeptides of the invention are used in processes forconverting an organic material, e.g., a biomass, such as alignocellulosic biomass, to a biofuel, such as a bioethanol, biobutanol,biomethanol, a biopropanol, or otherwise are used in processes forhydrolyzing or digesting biomaterials such that they can be used as abiofuel (including biodiesel or bioethanol, biobutanol, biomethanol orbiopropanol), or for making it easier for the biomass to be processedinto a fuel. In an alternative aspect, polypeptides of the invention areused in processes for a transesterification process reacting an alcohol(like methanol, butanol, propanol, ethanol) with a triglyceride oilcontained in a vegetable oil, animal fat or recycled greases, formingfatty acid alkyl esters (biodiesel) and glycerin. In one aspect,biodiesel is made from soybean oil or recycled cooking oils. Animal'sfats, other vegetable oils, and other recycled oils can also be used toproduce biodiesel, depending on their costs and availability. In anotheraspect, blends of all kinds of fats and oils are used to produce abiodiesel fuel of the invention.

Enzymes of the invention can also be used in glycerin refining. Theglycerin by-product contains unreacted catalyst and soaps that areneutralized with an acid. Water and alcohol are removed to produce 50%to 80% crude glycerin. The remaining contaminants include unreacted fatsand oils, which can be processes using the polypeptides of theinvention. In a large biodiesel plants of the invention, the glycerincan be further purified, e.g., to 99% or higher purity, for thepharmaceutical and cosmetic industries.

Fuels (including bioalcohols such as bioethanols, biomethanols,biobutanols or biopropanols, or biodiesels) made using the polypeptidesof the invention, including the mixture of enzymes or “cocktails” of theinvention, can be used with fuel oxygenates to improve combustioncharacteristics. Adding oxygen results in more complete combustion,which reduces carbon monoxide emissions. This is another environmentalbenefit of replacing petroleum fuels with biofuels (e.g., a fuel of theinvention). A biofuel made using the compositions and/or methods of thisinvention can be blended with gasoline to form an E10 blend (about 5% to10% ethanol and about 90% to 95% gasoline), but it can be used in higherconcentrations such as E85 or in its pure form. A biofuel made using thecompositions and/or methods of this invention can be blended withpetroleum diesel to form a B20 blend (20% biodiesel and 80% petroleumdiesel), although other blend levels can be used up to B100 (purebiodiesel).

In one aspect, the polypeptides of this invention are used in processesfor converting organic material, e.g., a biomass, such as alignocellulosic biomass, to methanol, butanol, propanol and/or ethanol.The invention also provides processes for making ethanol (“bioethanol”)methanol, butanol and/or propanol from compositions comprising organicmaterial, e.g., a biomass, such as a lignocellulosic biomass. Theorganic material, e.g., a biomass, such as a lignocellulose biomassmaterial, can be obtained from agricultural crops, as a byproduct offood or feed production, or as biomass waste products, such as plantresidues and waste paper or construction and/or demolition wastes ordebris. Examples of suitable plant residues for treatment withpolypeptides of the invention include grains, seeds, stems, leaves,hulls, husks, corn cobs, corn stover, straw, grasses (e.g., Indiangrass, such as Sorghastrum nutans; or, switch grass, e.g., Panicumspecies, such as Panicum virgatum), and the like, as well as wood, woodchips, wood pulp, and sawdust. Examples of paper waste suitable fortreatment with polypeptides of the invention include discard photocopypaper, computer printer paper, notebook paper, notepad paper, typewriterpaper, and the like, as well as newspapers, magazines, cardboard, andpaper-based packaging materials. Examples of construction and demolitionwastes and debris include wood, wood scraps, wood shavings and sawdust.

In one aspect, the enzymes and methods of the invention can be used inconjunction with more “traditional” means of making methanol, butanol,propanol and/or ethanol from biomass, e.g., as methods comprisinghydrolyzing biomass (e.g., lignocellulosic materials) by subjectingdried biomass material in a reactor to a catalyst comprised of a dilutesolution of a strong acid and a metal salt; this can lower theactivation energy, or the temperature, of cellulose hydrolysis to obtainhigher sugar yields; see, e.g., U.S. Pat. Nos. 6,660,506; 6,423,145.

Another exemplary method that incorporated use of enzymes of theinvention comprises hydrolyzing biomass (e.g., lignocellulosicmaterials) containing xylan, hemicellulose, cellulose and/or lignin bysubjecting the material to a first stage hydrolysis step in an aqueousmedium at a temperature and a pressure chosen to effect primarilydepolymerization of hemicellulose without major depolymerization ofcellulose to glucose. This step results in a slurry in which the liquidaqueous phase contains dissolved monosaccharides resulting fromdepolymerization of hemicellulose and a solid phase containing celluloseand lignin. A second stage hydrolysis step can comprise conditions suchthat at least a major portion of the cellulose is depolymerized, suchstep resulting in a liquid aqueous phase containing dissolved/solubledepolymerization products of cellulose. See, e.g., U.S. Pat. No.5,536,325. Enzymes of the invention can be added at any stage of thisexemplary process.

Another exemplary method that incorporated use of enzymes of theinvention comprises processing a biomass material by one or more stagesof dilute acid hydrolysis with about 0.4% to 2% strong acid; andtreating an unreacted solid lignocellulosic component of the acidhydrolyzed biomass material by alkaline delignification to produceprecursors for biodegradable thermoplastics and derivatives. See, e.g.,U.S. Pat. No. 6,409,841. Enzymes of the invention can be added at anystage of this exemplary process.

Another exemplary method that incorporated use of enzymes of theinvention comprises prehydrolyzing biomass (e.g., lignocellulosicmaterials) in a prehydrolysis reactor; adding an acidic liquid to thesolid lignocellulosic material to make a mixture; heating the mixture toreaction temperature; maintaining reaction temperature for timesufficient to fractionate the lignocellulosic material into asolubilized portion containing at least about 20% of the lignin from thelignocellulosic material and a solid fraction containing cellulose;removing a solubilized portion from the solid fraction while at or nearreaction temperature wherein the cellulose in the solid fraction isrendered more amenable to enzymatic digestion; and recovering asolubilized portion. See, e.g., U.S. Pat. No. 5,705,369. Enzymes of theinvention can be added at any stage of this exemplary process.

The invention provides methods for making motor fuel compositions (e.g.,for spark ignition motors) based on liquid hydrocarbons blended with afuel grade alcohol made by using an enzyme or a method of the invention.In one aspect, the fuels made by use of an enzyme of the inventioncomprise, e.g., coal gas liquid- or natural gas liquid-ethanol blends.In one aspect, a co-solvent is biomass-derived 2-methyltetrahydrofuran(MTHF). See, e.g., U.S. Pat. No. 6,712,866.

In one aspect, methods of the invention for the enzymatic degradation ofbiomass (e.g., lignocellulosic materials), e.g., for production of abiofuel, e.g., an ethanol, from a biomass or any organic material, canalso comprise use of ultrasonic treatment of a biomass material; see,e.g., U.S. Pat. No. 6,333,181.

In another aspect, methods of the invention for producing a biofuel,e.g., an ethanol (a bioethanol) from a biomass (e.g., a cellulosic)substrate comprise providing a reaction mixture in the form of a slurrycomprising biomass (e.g., a cellulosic) substrate, an enzyme of thisinvention and a fermentation agent (e.g., within a reaction vessel, suchas a semi-continuously solids-fed bioreactor), and the reaction mixtureis reacted under conditions sufficient to initiate and maintain afermentation reaction (as described, e.g., in U.S. Pat. App. No.20060014260). In one aspect, experiment or theoretical calculations candetermine an optimum feeding frequency. In one aspect, additionalquantities of the biomass (e.g., a cellulosic) substrate and the enzymeare provided into the reaction vessel at an interval(s) according to theoptimized feeding frequency.

One exemplary process for making a biofuels and biodiesels of theinvention is described in U.S. Pat. App. Pub. Nos. 20050069998;20020164730; and in one aspect comprises stages of grinding the biomass(e.g., lignocellulosic material) (e.g., to a size of 15-30 mm),subjecting the product obtained to steam explosion pretreatment (e.g.,at a temperature of 190-230° C.) for between 1 and 10 minutes in areactor; collecting the pre-treated material in a cyclone or relatedproduct of manufacture; and separating the liquid and solid fractions byfiltration in a filter press, introducing the solid fraction in afermentation deposit and adding one or more enzymes of the invention,and in one aspect, another enzyme is also added, e.g., a cellulaseand/or beta-glucosidase enzyme (e.g., dissolved in citrate buffer pH4.8).

Another exemplary process for making a biofuels and biodiesels of theinvention comprising methanol, butanol, propanol and/or ethanol usingenzymes of the invention comprises pretreating a starting materialcomprising a biomass (e.g., a lignocellulosic) feedstock comprising atleast a xylan, a hemicellulose and/or a cellulose. In one aspect, thestarting material comprises potatoes, soybean (rapeseed), barley, rye,corn, oats, wheat, beets or sugar cane or a component or waste or foodor feed production byproduct. The starting material (“feedstock”) isreacted at conditions which disrupt the plant's fiber structure toeffect at least a partial hydrolysis of the biomass (e.g., hemicelluloseand/or cellulose). Disruptive conditions can comprise, e.g., subjectingthe starting material to an average temperature of 180° C. to 270° C. atpH 0.5 to 2.5 for a period of about 5 seconds to 60 minutes; or,temperature of 220° C. to 270° C., at pH 0.5 to 2.5 for a period of 5seconds to 120 seconds, or equivalent. This generates a feedstock withincreased accessibility to being digested by an enzyme, e.g., acellulase enzyme of the invention. U.S. Pat. No. 6,090,595.

Exemplary conditions for hydrolysis of biomass (e.g., a lignocellulosicmaterial) by an enzyme of this invention include reactions attemperatures between about 30° C. and 48° C., and/or a pH between about4.0 and 6.0. Other exemplary conditions include a temperature betweenabout 30° C. and 60° C. and a pH between about 4.0 and 8.0.

Biofuels and Biologically Produced Alcohols

The invention provides biofuels and synthetic fuels, including liquidsand gases (e.g., syngas) and biologically produced alcohols, and methodsfor making them, using the compositions (e.g., enzyme and nucleic acids,and transgenic plants, animal, seeds and microorganisms) and methods ofthe invention. The invention provides biofuels and biologically producedalcohols comprising enzymes, nucleic acids, transgenic plants, animals(e.g., microorganisms, such as bacteria or yeast) and/or seeds of theinvention. In one aspect, these biofuels and biologically producedalcohols are produced from a biomass.

The invention provides biologically produced alcohols, such as ethanol,methanol, propanol and butanol produced by methods of the invention,which include the action of microbes and enzymes of the inventionthrough fermentation (hydrolysis) to result in an alcohol fuel.

Biofuels as a Liquid or a Gas Gasoline

The invention provides biofuels and synthetic fuels in the form of agas, or gasoline, e.g., a syngas. In one aspect, methods of theinvention comprising use of enzymes of the invention for chemical cyclesfor natural biomass conversion, e.g., for the hydrolysis of a biomass tomake a biofuel, e.g., a bioethanol, biopropanol, bio-butanol or abiomethanol, or a synthetic fuel, in the form of a liquid or as a gas,such as a “syngas”.

For example, invention provides methods for making biofuel gases andsynthetic gas fuels (“syngas”) comprising a bioethanol, biopropanol,bio-butanol and/or a biomethanol made using a polypeptide of theinvention, or made using a method of the invention; and in one aspectthis biofuel gas of the invention is mixed with a natural gas (can alsobe produced from biomass), e.g., a hydrogen or a hydrocarbon-based gasfuel.

In one aspect, the invention provides methods for processing biomass toa synthetic fuel, e.g., a syngas, such as a syngas produced from abiomass by gasification. In one aspect, the invention provides methodsfor making an ethanol, propanol, butanol and/or methanol gas from asugar cane, e.g., a bagasse. In one aspect, this fuel, or gas, is usedas motor fuel, e.g., an automotive, truck, airplane, boat, small engine,etc. fuel. In one aspect, the invention provides methods for making anethanol, propanol, butanol and/or methanol from a plant, e.g., corn, ora plant product, e.g., hay or straw (e.g., a rice straw or a wheatstraw, or any the dry stalk of any cereal plant), or an agriculturalwaste product. Cellulosic ethanol, propanol, butanol and/or methanol canbe manufactured from a plant, e.g., corn, or plant product, e.g., hay orstraw, or an agricultural waste product (e.g., as processed by IogenCorporation of Ontario, Canada).

In one aspect, the ethanol, propanol, butanol and/or methanol made usinga method of composition of the invention can be used as a fuel (e.g., agasoline) additive (e.g., an oxygenator) or in a direct use as a fuel.For example, a ethanol, propanol, butanol and/or methanol, including afuel, made by a method of the invention can be mixed with ethyl tertiarybutyl ether (ETBE), or an ETBE mixture such as ETBE containing 47%ethanol as a biofuel, or with MTBE (methyl tertiary-butyl ether). Inanother aspect, a ethanol, propanol, butanol and/or methanol, includinga fuel, made by a method of the invention can be mixed with:

IUPAC name Common name but-1-ene α-butylene cis-but-2-ene cis-β-butylenetrans-but-2-ene trans-β-butylene 2-methylpropene isobutylene

A butanol and/or ethanol made by a method of the invention (e.g., usingan enzyme of the invention) can be further processed using “A.B.E.”(Acetone, Butanol, Ethanol) fermentation; in one aspect, butanol beingthe only liquid product. In one aspect, this butanol and/or ethanol isburned “straight” in existing gasoline engines (without modification tothe engine or car), produces more energy and is less corrosive and lesswater soluble than ethanol, and can be distributed via existinginfrastructures.

The invention also provides mixed alcohols wherein one, several or allof the alcohols are made by processes comprising at least one method ofthe invention (e.g., using an enzyme of the invention), e.g., comprisinga mixture of ethanol, propanol, butanol, pentanol, hexanol, andheptanol, such as ECALENE™ (Power Energy Fuels, Inc., Lakewood, Colo.),e.g.:

Exemplary Fuel of the Invention Component Weight % Methanol 0% Ethanol75%  Propanol 9% Butanol 7% Pentanol 5% Hexanol & Higher 4%

In one aspect, one, several or all of these alcohols are made by aprocess of the invention using an enzyme of the invention, and theprocess can further comprise a biomass-to-liquid technology, e.g., agasification process to produce syngas followed by catalytic synthesis,or by a bioconversion of biomass to a mixed alcohol fuel.

The invention also provides processes comprising use of an enzyme of theinvention incorporating (or, incorporated into) “gas to liquid”, or GTL;or “coal to liquid”, or CTL; or “biomass to liquid” or BTL; or “oilsandsto liquid”, or OTL, processes; and in one aspect these processes of theinvention are used to make synthetic fuels. In one aspect, one of theseprocesses of the invention comprises making a biofuel (e.g., a synfuel)out of a biomass using, e.g., the so-called “Fischer Tropsch” process (acatalyzed chemical reaction in which carbon monoxide and hydrogen areconverted into liquid hydrocarbons of various forms; typical catalystsused are based on iron and cobalt; the principal purpose of this processis to produce a synthetic petroleum substitute for use as syntheticlubrication oil or as synthetic fuel). In one aspect, this syntheticbiofuel of the invention can contain oxygen and can be used as additivein high quality diesel and petrol.

In alternative aspects, the processes of the invention use variouspretreatments, which can be grouped into three categories: physical,chemical, and multiple (physical+chemical). Any chemicals can be used asa pretreatment agent, e.g., acids, alkalis, gases, cellulose solvents,alcohols, oxidizing agents and reducing agents. Among these chemicals,alkali is the most popular pretreatment agent because it is relativelyinexpensive and results in less cellulose degradation. The commonalkalis sodium hydroxide and lime also can be used as pretreatmentagents. Although sodium hydroxide increases biomass digestibilitysignificantly, it is difficult to recycle, is relatively expensive, andis dangerous to handle. In contrast, lime has many advantages: it issafe and very inexpensive, and can be recovered by carbonating washwater with carbon dioxide.

In one aspect, the invention provides a multi-enzyme system (includingat least one enzyme of this invention) that can hydrolyzepolysaccharides in a biomass, e.g. sugarcane, e.g., bagasse, a componentof sugarcane processed in sugar mills. In one aspect, the biomass isprocessed by an enzyme of the invention made by an organism (e.g.,transgenic animal, plants, transformed microorganism) and/or byproduct(e.g., harvested plant, fruit, seed) expressing an enzyme of theinvention. In one aspect, the enzyme is a recombinant enzyme made by theplant or biomass which is to be processed to a fuel, e.g., the inventionprovides a transgenic sugarcane bagasse comprising an enzyme of theinvention. In one aspect, these compositions and products used inmethods of the invention comprising chemical cycles for natural biomassconversion, e.g., for the hydrolysis of a biomass to make a biofuel,e.g., bioethanol, biopropanol, bio-butanol, biomethanol, a syntheticfuel in the form of a liquid or a gas, such as a “syngas”.

In one aspect, the invention provides a biofuel, e.g., a biogas,produced by the process of anaerobic digestion of organic material byanaerobes, wherein the process comprises use of an enzyme of theinvention or a method of the invention. This biofuel, e.g., a biogas,can be produced either from biodegradable waste materials or by the useof energy crops fed into anaerobic digesters to supplement gas yields.The solid output, digestate, can also be used as a biofuel.

In one aspect, the invention provides a biofuel, e.g., a biogas,comprising a methane, wherein the process comprises use of an enzyme ofthe invention or a method of the invention. This biofuel, e.g., abiogas, can be recovered in industrial anaerobic digesters andmechanical biological treatment systems. Landfill gas can be furtherprocessed using an enzyme of this invention or a process of thisinvention; before processing landfill gas can be a less clean form ofbiogas produced in landfills through naturally occurring anaerobicdigestion. Paradoxically if landfill gas is allowed to escape into theatmosphere it is a potent greenhouse gas.

The invention provides methods for making biologically produced oils andgases from various wastes, wherein the process comprises use of anenzyme of the invention or a method of the invention. In one aspect,these methods comprise thermal depolymerization of waste to extractmethane and other oils similar to petroleum; or, e.g., a bioreactorsystem that utilizes nontoxic photosynthetic algae to take insmokestacks flue gases and produce biofuels such as biodiesel, biogasand a dry fuel comparable to coal, e.g., as designed by GreenFuelTechnologies Corporation, of Cambridge, Mass.

The invention provides methods for making biologically produced oils,including crude oils, and gases that can be used in diesel engines,wherein the process comprises use of an enzyme of the invention or amethod of the invention. In one aspect, these methods can refinepetroleum, e.g., crude oils, into kerosene, pertroleum, diesel and otherfractions.

The invention provides methods (using an enzyme of the invention or amethod of the invention) for making biologically produced oils from:

-   -   Straight vegetable oil (SVO),    -   Waste vegetable oil (WVO)—waste cooking oils and greases        produced in quantity mostly by commercial kitchens.    -   Biodiesel obtained from transesterification of animal fats and        vegetable oil, directly usable in petroleum diesel engines.    -   Biologically derived crude oil, together with biogas and carbon        solids via the thermal depolymerization of complex organic        materials including non oil based materials; for example, waste        products such as old tires, offal, wood and plastic.    -   Pyrolysis oil; which may be produced out of biomass, wood waste        etc. using heat only in the flash pyrolysis process (the oil may        have to be treated before using in conventional fuel systems or        internal combustion engines).    -   Wood, charcoal, and dried dung.

Medical and Research Applications

Xylanases of the invention, including the enzyme mixtures or “cocktails”of the invention, can be used as antimicrobial agents due to theirbacteriolytic properties. Xylanases of the invention can be used toeliminating or protecting animals from salmonellae, as described ine.g., PCT Application Nos. WO0049890 and WO9903497. In another aspect ofthe invention, the xylanases of the invention can also be used anantimicrobial surface cleanser or microbial repellent.

Other Industrial and Medical Applications

As discussed above, xylanases of the invention, including the enzymemixtures or “cocktails” of the invention, can be used can be used, e.g.,in a wide variety of industrial processes, medical and research(laboratory) applications, and food, animal feed and beverageapplications. New xylanases are discovered by screening existinglibraries and DNA libraries constructed from diverse mesophilic andmoderately thermophilic locations as well as from targeted sourcesincluding digestive flora, microorganisms in animal waste, soil bacteriaand highly alkaline habitats. Biotrap and primary enrichment strategiesusing arabinoxylan substrates and/or non-soluble polysaccharidefractions of animal feed material are also useful.

Two screening formats (activity-based and sequence-based) are used inthe discovery of novel xylanases. The activity-based approach is directscreening for xylanase activity in agar plates using a substrate such asazo-xylan (Megazyme). Alternatively a sequence-based approach may beused, which relies on bioinformatics and molecular biology to designprobes for hybridization and biopanning. See, for example, U.S. Pat.Nos. 6,054,267, 6,030,779, 6,368,798, 6,344,328. Hits from the screeningare purified, sequenced, characterized (for example, determination ofspecificity, temperature and pH optima), analyzed using bioinformatics,subcloned and expressed for basic biochemical characterization. Thesemethods may be used in screening for xylanases useful in a myriad ofapplications, including dough conditioning and as animal feed additiveenzymes.

In characterizing enzymes obtained from screening, the exemplary utilityin dough processing and baking applications may be assessed.Characterization may include, for example, measurement of substratespecificity (xylan, arabinoxylan, CMC, BBG), temperature and pHstability and specific activity. A commercial enzyme may be used as abenchmark. In one aspect, the enzymes of the invention have significantactivity at pH≧7 and 25-35° C., are inactive on insoluble xylan, arestable and active in 50-67% sucrose.

In another aspect, utility as feed additives may be assessed fromcharacterization of candidate enzymes. Characterization may include, forexample, measurement of substrate specificity (xylan, arabinoxylan, CMC,BβG), temperature and pH stability, specific activity and gastricstability. In one aspect the feed is designed for a monogastric animaland in another aspect the feed is designed for a ruminant animal. In oneaspect, the enzymes of the invention have significant activity at pH 2-4and 35-40° C., a half-life greater than 30 minutes in gastric fluid,formulation (in buffer or cells) half-life greater than 5 minutes at 85°C. and are used as a monogastric animal feed additive. In anotheraspect, the enzymes of the invention have one or more of the followingcharacteristics: significant activity at pH 6.5-7.0 and 35-40° C., ahalf-life greater than 30 minutes in rumen fluid, formulation stabilityas stable as dry powder and are used as a ruminant animal feed additive.

Enzymes are reactive toward a wide range of natural and unnaturalsubstrates, thus enabling the modification of virtually any organic leadcompound. Moreover, unlike traditional chemical catalysts, enzymes arehighly enantio- and regio-selective. The high degree of functional groupspecificity exhibited by enzymes enables one to keep track of eachreaction in a synthetic sequence leading to a new active compound.Enzymes are also capable of catalyzing many diverse reactions unrelatedto their physiological function in nature. For example, peroxidasescatalyze the oxidation of phenols by hydrogen peroxide. Peroxidases canalso catalyze hydroxylation reactions that are not related to the nativefunction of the enzyme. Other examples are xylanases which catalyze thebreakdown of polypeptides. In organic solution some xylanases can alsoacylate sugars, a function unrelated to the native function of theseenzymes.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds. Eachbiocatalyst is specific for one functional group, or several relatedfunctional groups and can react with many starting compounds containingthis functional group. The biocatalytic reactions produce a populationof derivatives from a single starting compound. These derivatives can besubjected to another round of biocatalytic reactions to produce a secondpopulation of derivative compounds. Thousands of variations of theoriginal compound can be produced with each iteration of biocatalyticderivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so-called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies and compounds can be synthesizedand tested free in solution using virtually any type of screening assay.It is important to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods. (For further teachings on modification ofmolecules, including small molecules, see PCT/US94/09174).

In one aspect, the invention provides a composition comprising at leastone mucoadhesive polymer that is capable of forming a hydrogel and atone least water soluble polymer, and one or more enzymes of theinvention. This formulation can be used in any industrial, food or feedprocessing or medical or research application of the invention, i.e.,any application using an enzyme or nucleic acid of the invention. In oneaspect, the formulation forms a hydrogel in aqueous solution that hasmucoadhesive properties; this can be capable of releasing enzymes,microorganisms capable of generating enzymes of the invention, orantibodies of the invention, over an extended period of time.Alternatively, the hydrogel can entrap enzymes, microorganisms capableof generating enzymes of the invention, or antibodies of the inventionand release them over a defined (e.g., an extended) period of time.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Xylanase Assay with Wheat Arabinoxylan as Substrate

The following example describes an exemplary xylanase assay that can beused, for example, to determine if an enzyme is within the scope of theinvention. Enzymes of the invention, e.g., SEQ ID NO:2 having one ormore amino acid residue changes (mutations) as set forth in Table 1 andas described herein, also include a genus of polypeptides having varioussequence identities based on the exemplary SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24, can besubjected to an assay at pH 8 (Na-phosphate buffer) and 70° C. usingwheat arabinoxylan as a substrate.

Example 2 Determination of Melting Temperature and Xylanase ActivityDifferential Scanning Calorimetry (DSC)

The melting temperature transition midpoint (T_(m)) for each enzyme ofthe invention, e.g. SEQ ID NO:2 having one or more amino acid residuechanges (mutations) as set forth in Table 1 and as described herein, canbe determined by Differential Scanning calorimetry (DSC). Baselinesubtracted DSC data can be normalized for protein concentration.

In one assay, calorimetry can be performed using a Model 6100 NANO IIDSC™ apparatus (calorimetry Sciences Corporation, American Fork, Utah)using the DSCRUN™ (DSCRun) software package for data acquisition,CPCALC™ (CpCalc) for analysis, CPCONVERT™ (CpConvert) for conversioninto molar heat capacity from microwatts and CPDECONVOLUTE™(CpDeconvolute) for deconvolution. Analysis can be carried out with 1mg/ml recombinant protein in 20 mM potassium phosphate (pH 7.0) and 100mM KCl at a scan rate of 1° C./min. A constant pressure of 5 atm can bemaintained during all DSC experiments to prevent possible degassing ofthe solution on heating. The instrumental baseline can be recordedroutinely before the experiments with both cells filled with buffer.Reversibility of the thermally induced transitions can be tested byreheating the solution in the calorimeter cell immediately after coolingthe first run.

Alternatively, DSC measurements can be made using a VP-DSCmicrocalorimeter (Micro-Cal) in duplicate. In one aspect, a requiredsample volume is 540 μL. The concentrations of the protein can bebetween 0.1 to 0.5 mg/mL in 50 mM HEPES, pH 7.2; a sample of thedialysis buffer can be retained for base line controls. Each sample canbe heated from 40° C. to 110° C. Samples and/or buffer can be heated andcooled at a scan rate of 90° C./h. Buffer baselines were recordedmultiple times until the system reached a stable state. The T_(m) valuewas the temperature where maximum heat was released.

Xylanase Activity Assays

Enzymatic activities can be determined using 400 μL of 2% Azo-xylan assubstrate in 550 μL of CP (citrate-phosphate) buffer, pH 6.0 at theindicated temperatures. Activity measurements as a function of pH can bedetermined using 50 mM Britton and Robinson buffer solutions (pH 3.0,5.0, 6.0, 7.0, 8.0 and 9.0) prepared by mixing solutions of 0.1 Mphosphoric acid solution, 0.1 M boric acid and 0.1 M acetic acidfollowed by pH adjustment with 1 M sodium hydroxide. Reactions can beinitiated by adding 50 μL of 0.1 mg/ml of purified enzyme. Time pointscan be taken from 0 to 15 minutes where 50 μL of reaction mixture areadded to 200 μL of precipitation solution (100% ethanol). When all timepoints have been taken, samples are mixed, incubated for 10 minutes andcentrifuged at 3000 g for 10 minutes at 4° C. Supernatant (150 μL) canbe aliquoted into a fresh 96 well plate and absorbance is measured at590 nm. A590 values can be plotted against time and the initial rate isdetermined from the slope of the line.

Polysaccharide Fingerprinting.

Polysaccharide fingerprints can be determined by polysaccharide analysisusing carbohydrate gel electrophoresis (PACE). Beechwood xylan (0.1mg/mL, 100 μL, Sigma, Poole, Dorset, UK) or xylooligosaccharides (1 mM,20 μL, Megazyme, Wicklow, Ireland) can be treated with enzyme (1-3 μg)in a total volume of 250 μL for 16 hours. The reaction is buffered in0.1 M ammonium acetate pH 5.5. Controls without substrates or enzymesare performed under the same conditions to identify any unspecificcompounds in the enzymes, polysaccharides/oligosaccharides or labelingreagents. The reactions are stopped by boiling for 20 min. Assays can beindependently performed at least 2 times for each condition.Derivatization using ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid,Molecular Probes, Leiden, The Netherlands), electrophoresis and imagingare carried out as described (Goubet, F., Jackson, P., Deery, M. andDupree, P. (2002) Anal. Biochem. 300, 53-68).

Fitness Calculation.

The fitness (F_(n)), for a given enzyme variant, n, can be calculated byequally weighting increase in denaturation temperature transitionmidpoint (T_(m)) and increase (or decrease) in enzymatic activityrelative to the largest difference in each parameter across allvariants: F_(n)=F_(Tn)+F_(Vn), where F_(Tn)=T_(m) fitness factor of thevariant and F_(Vn)=activity fitness factor of the variant. The fitnessfactors for each (T_(m) and activity) are relative to the largestdifference in T_(m) or rate across all of the variants.F_(Tn)=(T_(m)−T_(mL))/(T_(mH)−T_(mL)) where T_(mH) is the T_(m) for thegiven variant, n, and T_(mL) is the lowest T_(m) across all variants andT_(mH) the highest T_(m) across all variants andF_(Vn)=(V_(n)−V_(L))/(V_(H)−V_(L)) where V_(n) is the relative rate forthe given variant, n, and V_(L) is the lowest rate across all variantsand V_(H) the highest rate across all variants.

Example 3 Pre-Treating Paper Pulp with Xylanases of the Invention

In one aspect, xylanases of the invention are used to treat/pretreatpaper pulp, or recycled paper or paper pulp, waste wood or wood chips,and the like. In one aspect, enzyme(s) of the invention are used toincrease the “brightness” of the paper via their use intreating/pretreating paper pulp, or recycled paper or paper pulp, andthe like.

In one aspect, xylanases of the invention are used to treat/pretreatpaper pulp, or recycled paper or paper pulp, and the like to reduce theKappa number. Kappa number is defined as a numerical value indicating apaper's relative lignin content—the higher the Kappa number, the higherthe lignin content. In some aspects, reduction in Kappa # has benefitswhen treating unbleached pulp (kappa #70-90), when then is used for,e.g., processing, such as in board manufacture. In some aspects, areduction in Kappa across the X stage allows lower alkali use in cookingor cooking to a higher target Kappa #. In some aspects, this results inhigher pulp strength, less machine refining and higher machine speeds.In some aspects, such results are seen using digester additives(surfactants) in linerboard mills; this can allow for better liquorpenetration, and allow lower effective alkali charge leading to higherpulp strength, lower refining and a 200 fpm (feet per minute) increasein machine speed.

This example describes an exemplary routine screening protocol todetermine whether a xylanase is useful in pretreating paper pulp; e.g.,in reducing the use of bleaching chemicals (e.g., chlorine dioxide,ClO₂) when used to pretreat Kraft paper pulp.

The screening protocol has two alternative test parameters: Impact ofxylanase treatment after an oxygen delignification step (post-O₂ pulp);and, impact of xylanase in a process that does not include oxygendelignification (pre-O₂ brownstock).

The invention provides pulp or paper treatment conditions that simulateprocess conditions in industrial situations, e.g., factories: forexample, at about pH 8.0; 70° C.; 60 min duration. For example, anexemplary process of the invention is schematically depicted in the FlowDiagram of FIG. 5; see also FIG. 6. However, the conditions of a processof method of the invention can be adjusted to any temperature, timeduration and/or pH, depending on the exemplary enzyme(s) of theinvention used and the objective of the process; for example, there area variety of ways to adjust pH in the various pulp and paper processesof the invention:

-   -   adding acid and/or base:        -   Hydrochloric acid (HCl)        -   d Sodium hydroxide (NaOH)        -   H₂ SO₄ (sulfuric acid)        -   NaHSO₄ (sodium hydrogen sulfate)        -   H₂SO₃ (sulfurous acid)        -   H3PO₄ (phosphoric acid)        -   HF (hydrofluoric acid)        -   CH3CO₂H (acetic acid)        -   H₂CO₃ (carbonic acid)        -   H₂S (hydrogen sulfide)        -   NaH₂PO₄ (sodium dihydrogen phosphate)        -   NH₄Cl (ammonium chloride)        -   HCN (hydrocyanic acid)        -   Na₂SO₄ (sodium sulfate)        -   NaCl (sodium chloride)        -   NaCH₃CO₂ (sodium acetate)        -   NaHCO₃ (sodium bicarbonate)        -   Na₂HPO₄ (sodium hydrogen phosphate)        -   Na₂S O₃ (sodium sulfite)        -   NaCN (sodium cyanide)        -   NH₃ (aqueous ammonia)        -   Na₂CO₃ (sodium carbonate)        -   Na₃PO₄ (sodium phosphate)    -   bubbling in gas, e.g. CO₂ (which forms an acid with water when        dissolved)

Dose Response Determination for Xylanases on Pre-02 Brownstock

Conditions for xylanase stage (X-stage) as follows:

pH 8

Temperature 70° C.

Time 60 min

Kappa factor 0.24

For no-enzyme control, kappa factor was 0.30

Pretreatment of Intercontinental Pre-O₂ Brownstock Xylanase

Determination of ClO₂ Dose Response in D_(o)

Experimental outline

Pre-O₂ Brownstock

Initial kappa 31.5

X stage conditions

Xylanase charge 0.7 U/gm

Temperature 70° C.

pH 8

Treatment time 1 hr

Pulp consistency 10%

Bleach sequence XDE_(p)

Kappa factor 0.22, 0.26 and 0.30 (% D on pulp: 2.63, 3.12 and 3.60)

Determination of ClO₂ Dose Response in D_(o):

Xylanase 0.7 U/g, pH 8.0, 70° C., 1 hr

Pulp: Pre-02 Brownstock, initial kappa 31.5

Percentage saving of ClO₂ is of little significance to the industry.Their primary concern is lbs of ClO₂ required per ton OD pulp. Thismakes sense when one considers that a lower percentage saving seen witha high initial kappa brownstock can be more valuable in terms of lbs ofClO₂ saved than a higher percentage reduction for a low initial kappapulp which will require a lower total charge of ClO₂ to reach targetbrightness.

Relationship between Brightness, Yield and Kappa Factor for BleachedControl Pulp:

Bleaching with increasing doses of ClO₂ to achieve higher targetbrightness results in increased loss of pulp yield. This is an issuebecause pulp at this stage of the process has a value of almost $400 perton and loss of cellulose costs money.

A benefit of xylanase (e.g., a xylanase of the invention) is that use ofa lower ClO₂ dose can reduce yield losses as long as the action of thexylanase itself doesn't cancel out the gain.

Dose Response Data for Pretreatment of Pre-O₂ Brownstock with XylanaseExperimental outline

Northwood Pre-O₂ Brownstock

Initial kappa 28.0

Initial consistency 32.46%

Initial brightness 28.37

X stage conditions

Xylanase charge 0 to 2.70 U/gm

Temperature 58° C. to 61° C.

pH 8.2 to 8.5

Treatment time 1 hr

Bleach sequence XDE_(p)

Kappa factor 0.24

ClO₂ saving calculated for Kappa factors between 0.24 and 0.30

The purpose of this experiment is to evaluate xylanases on unwashed SPFbrownstock. Results can show dose-dependent increases in finalbrightness for pulp treated with XYLB (E.c), with brightness achieved inpresence of xylanase at lower Kf of 0.24, approaching brightnessachieved at higher Kf of 0.30 asymptotically.

Example 4 Novel Biobleaching Assay for Assessing Xylanase Performance inEnhancing the Brightness of Pulp

This example describes an exemplary protocol, a “biobleaching assay,”that can be used to determine if a polypeptide has xylanase activity andis within the scope of the invention. This assay can be used to assessthe performance of an exemplary enzyme of the invention, for example SEQID NO:2 having one or more amino acid residue changes (mutations) as setforth in Table 1 and as described herein, or a sequence having asequence identity (as described herein) to an exemplary SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ IDNO:24; in enhancing the brightness of a pulp, e.g., a Kraft Pulp.

The invention provides biobleaching procedures, e.g., a three-stagebiobleaching procedure that closely simulates the conditions of anactual pulp mill bleach plant, as illustrated in FIG. 5; including aprocess as illustrated in FIG. 6. This bleach sequence is designated by(X)DoEp, in which X represents the xylanase treatment stage (using,e.g., an enzyme of the invention), D for chlorine dioxide bleachingstage, and Ep for alkaline peroxide extraction stage. Many differentfeedstocks may be used, for example, Southern Softwood Kraft Brownstock(without oxygen delignification) and hardwood Kraft pulp (e.g., mapleand aspen). Upon completion of each biobleaching round, ensuing pulp canbe used to produce TAPPI (Technical Association of Pulp and PaperIndustries, the technical association for the worldwide pulp, paper andconverting industry)—standard handsheets. The GE % brightness of eachhandsheet can be measured, and the brightness values can be used as theindication of how well each enzyme performs on the pulp during theenzymatic pretreatment stage (X).

Pulp Biobleaching:

Pulp was bleached in 10-g batches in sealed plastic bags using a 3-stage(X)DoEp sequence, as illustrated in FIG. 5. The treatment conditions atthe three stages can be summarized as follows:

-   -   X stage: 10% (w/v) consistency at 65° C. and pH=8 for 60 min    -   Do stage: 4% (w/v) consistency at 60° C. for 30 min; Kappa        Factor=0.18 for enzyme treated samples, and 0.18 and 0.21 for        no-enzyme controls.    -   Ep stage: 10% (w/v) consistency at 75° C. for 90 min; caustic        charge: 1.7% (w NaOH/w OD pulp) and H₂O₂ charge: 0.5% (w/w)

As noted in FIG. 5, in one aspect, raw pulp is washed to reduce pH to pH8.5; pulp is filter pressed and divided into bags. At each stage, bagscan be incubated in a water bath at the desired temperature and each bagis taken out and kneaded thoroughly every 10 min to ensure uniform massand heat transfer within the pulp mass. After each treatment, pulp canbe filtered, washed with 2 L of DI water and filtered again beforereceiving the next treatment. The moisture content of the pulp can bemeasured using a Mettler-Toledo moisture analyzer (Fisher Scientific,USA).

As noted in FIG. 5, in one aspect, after the pulp is filter pressed anddivided into bags, in the X stage, the pulp can be resuspended, filterpressed, the pH adjusted; and then, incubated with enzyme at 10% solids,65° C., 1 hour; then kneaded for 10 minutes. At the Do stage the pulpcan be resuspended, washed, pH set to 4.0, and filter pressed; then,impregnated with ClO₂ at 4% solids (i.e., 4% (w/v) consistency) at 60°C. for 30 min; then kneaded for 10 minutes. At the Do stage the KappaFactor=0.18 for enzyme treated samples, and 0.18 and 0.21 for no-enzymecontrols. At the Ep stage the pulp can be resuspended, washed, andfilter pressed; then, incubated with NaOH and H₂ O₂ at 10% solids (i.e.,10% (w/v) consistency) at 75° C. for 90 min; then kneaded for 10minutes. The caustic charge: 1.7% (w NaOH/w OD pulp) and H₂O₂ charge:0.5% (w/w). After kneading, handsheets were formed.

Handsheets: As noted in FIG. 5, in one aspect, handsheets can be formed(4 m pulp, pH about 6.5); handsheets can be made from unbleached andbleached pulp using TAPPI standard equipment (Kalamazoo Paper Chemicals,Richland, Mich.) according to TAPPI method T-272 sp-97. The GE %brightness of each handsheet can be measured using a BRIGHTMETER MICROS-5/BC™ (Technidyne Corp., New Albany, Ind.) according to TAPPI methodT-452 om-98 (reference at 457 nm).

Example 5 Novel Biobleaching Process

This example describes a novel biobleaching process of the invention, asillustrated in FIG. 6. This process can be practiced using any xylanaseenzyme, including a polypeptide of the invention, which includes apolypeptide having at 50% to 99% or more sequence identity to anexemplary enzyme of the invention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and alsoincludes any polypeptide having the sequence of SEQ ID NO:2 having oneor more amino acid residue changes (mutations) as set forth in Table 1and as described herein.

This exemplary process of the invention can have a starting materialcomprising “brownstock,” which can be described as: 1) feedstockpreparation—logs coming into the paper mill are debarked, chipped andscreened to remove overthick chips, fines, knots and foreign matter, 2)pulping—wood chips are cooked at 160° C. to 190° C. under pressure forseveral hours in a concentrated liquor of sodium hydroxide and sodiumsulfide to separate cellulose fibers and increase cellulose content byextracting the majority of unwanted lignin. The output of this step isreferred to as “brownstock”.

This process of the invention comprises a “Bleaching Step”—a multistageprocess by which residual lignin and other chromophores are removed towhiten the pulp to target brightness in preparation for making paper orother products. Pulp is treated with oxidizing chemicals, for examplechlorine and chlorine dioxide, that attack lignin preferentially. In oneaspect the process comprises a bleaching sequence where pulp is reactedwith chlorine dioxide, the “D₀” stage (see also FIG. 5); extracted withalkali in the presence of hydrogen peroxide, the “Ep” stage (see alsoFIG. 5, the “Ep” stage); reacted with chlorine dioxide a second time, a“D1” stage; extracted with alkali and hydrogen peroxide, an Ep stage;and, reacted with chlorine dioxide a third time, a D2 stage. Inpracticing this process, bleaching can be subject to many variationswith respect to type and quantity of oxidizing chemicals used and thenumber of process steps (however, chlorine dioxide is currently the mostwidely used chemical oxidant). In one aspect, this process comprisespretreatment of cooked pulp with oxygen under pressure; the oxygenreactor can be at high pressure—at about 200 to 230° F. and pH 12 to 14(this is a common first step in bleaching, known as “oxygendelignification”).

In one aspect, this process comprises refining. For example, prior topapermaking bleached pulp is mechanically fined to collapse thecellulose fibers into flat ribbons, fibrilate their surfaces and improvetheir physical characteristics for papermaking. At any stage of theprocess following pulping, the pulp may be dewatered, washed andadjusted to a predetermined consistency by the addition of clean wateror recyled streams.

Xylanase (e.g., an enzyme of the invention) can be just added afterpulping, in the oxygen reactor or in the storage container just beforethe oxygen reactor. Xylanase (e.g., an enzyme of the invention) can beadded at multiple points (one or more or all points) in the bleachingprocess. In one aspect, a laccase is added to catalyze break-down oflignin. The laccase may be added at any stage of the process, includingin the oxygen reactor. Pulp may release various components thatself-mediate the laccase. Alternatively, in one aspect, organic orinorganic mediators can be added (see, e.g., DE 19723890 describing anoxidation system comprising an organic mediator and a laccase;alternative exemplary mediators include2,2′-azinobis(3-ethylbenzth-iazoline-5-sulphonate) (ABTS) as anexemplary organic mediator and potassium octacyanomolybdate [K₄Mo(CN)₈]as an exemplary inorganic mediator). Mediators as described in U.S.patent application no. 20030096394, can also be used in the processes ofthe invention, including any compound capable of enhancing theactivities of laccase and laccase-related enzymes.

In one aspect, an esterase, e.g. lipase, or oxidoreductase, e.g.peroxidase is added. In addition, pH and/or temperature can be modifiedin the reactor. In monitoring reactions of the invention, any lignincontent-measuring technique can be used, e.g., see U.S. PatentApplication No. 20020144795, describing a method to measure kappa numberor lignin content of kraft pulps based on the voltammetric measurementof catalytic reactions involving lignin and redox mediators.

Enzymes of the invention can also be used in with alkali-oxygenbleaching (oxygen delignification) processes as described, e.g., in U.S.Pat. No. 6,824,646, the process comprising bleaching lignocellulose pulpin aqueous alkali solution with oxygen and treating the pulp with ahemicellulase, while a liquid fraction delivered from the enzymetreatment step is separated from the hemicellulase treated reactionmixture, and subjected to a penetration treatment through a separationmembrane, for example, reverse osmosis membrane, to separate a permeatedfraction from a non-permeated fraction; and then the permeated fractionis fed to the alkali-oxygen bleaching (oxygen delignification) stepcomprising use of an enzyme of the invention.

In alternative aspects of this or any other process (method) of theinvention xylanases (e.g., enzymes of the invention) are used to reducebleaching chemicals, e.g., chlorine, chlorine dioxide, caustic,peroxide, or any combination thereof; and in alternative aspects, areduction of up to about 1%, 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 100%, ofchemicals can be seen in practicing the methods and using the enzymes ofthe invention. In one aspect, a 100% reduction in chemicals can beachieved when the xylanase is used in combination with a laccase orother enzyme, e.g., by use of enzyme cocktails; noting the inventionprovides enzyme mixtures, or “cocktails” comprising at least one enzymeof the invention and one or more other enzyme(s), which can be anotherxylanase, or any other enzyme.

In one aspect xylanases of the invention are used to reduce chlorinedioxide to allow recycling of water in the process; thus, there is lesswater used and less water dumped into the sewer. In one aspect xylanasesof the invention are used to allow more lignin-rich pulp to enter thebleaching plant, allowing for better pulp yield and better quality pulp(i.e., less destruction during the cooking process). In one aspect,xylanases of the invention are used to increase the overall brightnessof the paper. In one aspect, xylanases of the invention are used tolower the kappa number of the pulp.

Xylanases of the invention can be used, and the processes of theinvention can be practiced, on all wood types, including, for example,on hard wood with, e.g., oxygen delignification, hard wood withoutoxygen delignification, soft wood with oxygen delignification and softwood without oxygen delignification, and the like. Xylanases of theinvention can be used, and the processes of the invention can bepracticed for processing of recycled paper and/or pulp.

Oxygen delignification typically requires the addition of a reactiontower between a brownstock washer and a bleach plant. Typically, oxygenand sodium hydroxide are added to brownstock. Reduction of bleachingchemistry by 50% can be achieved in the bleaching process if preceded byoxygen delignification. Washing follows oxygen delignification; effluentcan be recovered or discharged. Ozone delignification can be used inplace of oxygen delignification.

Example 6 Novel Biobleaching Assay

This example describes assays that can demonstrate xylanase activity inpolypeptides of the invention, e.g., the exemplary polypeptides of theinvention, or enzymes of the invention, e.g., SEQ ID NO:2 having one ormore amino acid residue changes (mutations) as set forth in Table 1 andas described herein, also include a genus of polypeptides having varioussequence identities based on the exemplary SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 or SEQ ID NO:24.

These xylanase activity studies can be based on those described byNelson (1944) J. Biol. Chem. 153:375-380, “Reducing Sugar Assay forXylanase”; and, Somogyi (1952) J. Biol. Chem. 195:19-23. This“Nelson-Somogyi” assay can be used to determine units of activity; datafrom “Nelson-Somogyi” assays demonstrating xylanase activity inpolypeptides of the invention by determining units of activity is setforth, below.

Enzyme unit determinations also can be determined using theNelson-Somogyi assay. Biobleaching assays can be based on methods fromTAPPI ((Technical Association of Pulp and Paper Industries, see above).Below a description along with references to the TAPPI protocols.

Pulp: In one aspect, two batches of southern softwood Kraft brownstockare obtained, e.g., from the Department of Wood and Fiber Science atNorth Carolina State University (Raleigh, N.C.). The pulp Kappa Numberscan be determined, e.g., typically are or are between 21.4 or 29.7, asanalyzied using TAPPI method T-236 om-99; see e.g., TAPPI Test Methods(2000-2001, 2003 173).

Pulp biobleaching: Pulp can be pretreated with xylanase and bleached in10 g batches in sealed plastic bags using a 3-stage xylanase/chlorinedioxide/alkaline peroxide sequence: (X)DoEp (see explanation above). Thetreatment conditions at the three stages can be:

X stage: 10% (w/v) consistency at 65° C. and pH 8 for 60 min.

Do stage: 4% (w/v) consistency at 60° C. for 30 min; a Kappa Factor of0.18 was used for enzyme treated samples, and 0.18 and 0.21 forno-enzyme control samples. The concentration of chlorine dioxide usedduring the Do stage was calculated using equation (1):

$\begin{matrix}{{{ClO}_{2}\mspace{14mu} \%} = \frac{{KF} \times K\#}{2.63}} & (1)\end{matrix}$

Where ClO₂% is equal to g pure chlorine dioxide per 100 g oven-dried(OD) pulp

KF is the Kappa Factor and K# is the Kappa Number of the pulp asdetermined by TAPPI method T-236 om-99, TAPPI Test Methods (2000-2001,2003 173), Ep stage: 10% (w/v) consistency at 75° C. for 90 min; causticcharge is 1.7% on pulp (w/w) and H₂O₂ charge is 0.5% on pulp (w/w).

At each stage, replicate bags can be incubated in a water bath at thedesired temperature and then removed and kneaded thoroughly every 10 minto ensure uniform mass and heat transfer within the pulp mass. Aftereach stage, pulp can be filtered, e.g., through a Buchner funnel linedwith a hard polypropylene filter (297-micron mesh, Spectrum Labs, Ft.Lauderdale, Fla.). The filtrate can be recycled once to catch the fines,and the pulp cake can be washed, e.g., with 2 L of DI water. The pulpcake can then be resuspended, e.g., in 1.5 L of DI water and pH can beadjusted, e.g., to pH 8 and pH 4 prior to X and Do stages, respectively.The moisture content of the pulp can be measured using a Mettler-Toledomoisture analyzer (Fisher Scientific, USA).

Handsheets can be made from the bleached pulp using TAPPI standardequipment (Kalamazoo Paper Chemicals, Richland, Mich.) according toTAPPI method T-272 sp-97, TAPPI Test Methods (2000-2001, 2003 173). TheGE % brightness of each handsheet TAPPI Test Methods (2000-2001, 2003173) can be measured, e.g., using a Technidyne BRIGHTMETER MICRO S-5/BC™(Technidyne Corp., New Albany, Ind.) according to TAPPI method T-452om-98.

COMPONENTS used in assay (1) 1M NaOH 0.5M Sodium phosphate buffer pH 81% Arabinoxylan - (Megazyme #P-WAXYM) prepared according to themanufacturer's instructions Xylose - prepare standards 0.15 mM-2 mMusing D-xylose dissolved in H₂O 96 well PCR plate (Fisher 05 500-48) PCRplate seals Standard 96 well clear plates 1 mL tubes (E&K 671511-RC) forthe 96 well block Solution 1: 12 g K⁺/Na⁺ tartrate; 24 g Na₂CO₃; 16 gNaHCO₃; 144 g Na₂SO₄ in 800 mL H₂O Solution 2: 4 g CuSO₄*5H₂O; 36 gNa₂SO₄ in 200 mL H₂O Reagent A: Mix 4 volumes of solution 1 with 1volume of solution 2. Note-make fresh daily Reagent B: 25 g (NH4)₂MoO₄in 450 mL H₂O; add 21 mL conc. H₂SO₄, mix. Dissolve 3 g Na₂HAsO₄*7H₂O in25 mL dH₂O; mix with ammonium molybdate solution and incubatereagent at37° C. for 24-48 h. Store solution in a dark bottle i.e. away from lightat room temperature.

Procedure

1. Prepare reagent A

2. Pipet 5 uL of 1 M NaOH into each well of a 96 well PCR plate. Keepplate on ice.

3. Prepare reaction mixture. Alternatively, you can make a master mixfor multiple samples. Here is the 1× mix. Add to the 1 mL tubes andplace into the 96 well block.

-   -   a. 50 uL pH8 Na-phosphate buffer    -   b. 250 uL of 1% substrate (to make a final concentration of        0.5%)    -   c. 150 uL H₂O

4. Preheat reaction mixture to desired temperature for 3 minutes.

5. Dilute the 0.5 M phosphate buffer to 5 mM pH 8 and make enzymedilutions using this buffer.

6. Pipet 75 uL of diluted enzyme into a well of a 96 well microtiterplate

7. Pipet 50 uL of diluted enzyme into the 1 mL tube containing thereaction mix.

8. At the desired timepoint, pipet 50 uL from each reaction mixture intotubes containing the NaOH (the NaOH will raise the pH to 12, quenchingthe reaction).

9. Add 50 uL of each standard to separate tubes also containing NaOH.Standards are linear within the range of 0.25 mM xylose to 2.0 mM. Useat least 4 standards to generate the standard curve.

10. Add 50 uL of Reagent A to each well. Seal plate using the Microseal™‘A’Film.

11. Heat the plate for 20 min. at 100° C. in a PCR machine. Set themachine to cool down to 4° C. after heating the samples.

12. Add 50 uL of reagent B to each tube, mix.

13. Note a significant amount of CO₂ is formed after addition of reagentB. Care should be taken so sample does not contaminate adjacent wells.

14. Pipet 100 uL of each sample or standard into separate wells of a 96well microtiter plate.

15. Read plate at 560 nm.

16. Plot standard curve data and express standards as umoles of xylosei.e. 50 uL of 2.5 mM xylose is 0.125 μmoles of xylose.

17. Subtract buffer control from sample data for each timepoint and plotthe data

18. Divide timepoint curve slope value by the xylose standard curveslope value

19. Multiply by 10 (accounts for the 50 uL samples (1/10 of the totalassay volume)

20. Divide by the volume used in the assay (0.05) to get μmoles ofxylose released per min per mL of enzyme or U/mL of enzyme.

21. Divide this number by the protein concentration to get U/mg.

“Units of Activity” data from the “Nelson-Somogyi” assays can be used todetermine dosing in biobleaching assays (based on TAPPI methods).

As noted above, the enzymes and processes of the invention can also beused in conjunction with a second approach to enzymatic bleaching usingoxidative enzymes such as laccase and/or manganese peroxidase (MnP) todelignify pulp. In one aspect of this second approach, of these enzymes,laccase is preferred, because MnP requires hydrogen peroxide, manganese(II) ions and a chelator. Laccase can cause delignification of pulpunder slight oxygen pressure, but is considerably more effective whenmediators are added, as discussed above.

Catalyst improved delignification methods can also be used inconjunction with the methods of the invention, for example, polysulfideor anthraquinone. Anthraquinone is a pulping reaction catalyst which canincrease the speed of pulping, increase yield, and reduce pulpingchemical usage by up to 10%. It is possible to use both anthraquinoneand polysulfide together.

In one aspect, laccase is used in conjunction with the methods of theinvention, as discussed above. For example, laccase is used in an oxygenreactor in a process of the invention, where the laccase breaks down thelignin in the oxygen reactor. While pulp may release various componentsthat self-mediate the laccase, in one aspect organic or inorganicmediators are added (see discussion above, e.g., alternative exemplarymediators include 2,2′-azinobis(3-ethylbenzth-iazoline-5-sulphonate)(ABTS) as an exemplary organic mediator and potassium octacyanomolybdate[K₄Mo(CN)₈] as an exemplary inorganic mediator, or mediators asdescribed in U.S. patent application no. 20030096394). In one aspect,another hydrolase, such as an esterase (e.g., a lipase) and/or anoxidoreductase (e.g., a peroxidase) is also added. In alternativeaspects, pH and/or temperature are modified in the reactor.

Example 7 Studies Demonstrating the Enzymatic Activity of Enzymes of theInvention

This example describes studies demonstrating the enzymatic activity ofthe exemplary xylanase enzymes of the invention, which demonstrates thatpolypeptides of this invention, which includes a polypeptide having at50% to 99% or more sequence identity to an exemplary enzyme of theinvention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22 and/or SEQ ID NO:24, and also includes anypolypeptide having the sequence of SEQ ID NO:2 having one or more aminoacid residue changes (mutations) as set forth in Table 1 and asdescribed herein, have xylanase activity.

An exemplary assay for evaluating these xylanases:

1. Initial Screen—using an azo-xylan (solution-based) substrate

-   -   a. Enzymatic activity of enzymes can be determined by an        azo-xylan assay using MEGAZYME® substrate Birchwood Azo-xylan in        100 mM sodium phosphate, pH 8, according to manufacturer's        recommended assay protocol. The concentrations of enzyme samples        can be adjusted such that they had equal amounts of xylanase        activity at pH8.    -   b. The azo-xylan assay are then repeated with normalized samples        in 100 mM sodium borate buffer at pH 10.4.

2. Initial Screen—ENZ-CHEK ULTRA XYLANASE ASSAY KIT™

(Invitrogen)

-   -   a. Xylanase enzyme samples can be prepared in the same manner as        for the azo-xylan assay (section 1, above).    -   b. The level of enzymatic activity of enzymes can be measured by        employing commercially available assay kit, e.g., sold by        Invitrogen under the name ENZ-CHEK ULTRA XYLANASE ASSAY KIT™        (Product number E33650). The ENZ-CHEK™ kit substrate produces        fluorescent signal in the presence of xylanases, which can be        used to quantify xylanase activities using kit-supplied        standards. The protocol used for testing xylanase enzymes can be        slightly modified from any manufacturer-recommended protocol.        The modifications can primarily involve, e.g., testing xylanases        at different pH and temperature that what is recommended by the        manufacturer.

3. Secondary Screen—Exemplary Pulp Assays

-   -   a. The enzymes from azo-xylan assay can be tested for activity        on wheat arabinoxylan using, e.g., a Nelson-Somogyi assay as        already described herein. They can be then tested in a        laboratory scale bleaching assays to determine the amount of        chemical savings each can achieved for a given pulp type and        chlorine dioxide loading. The ones that meet desired performance        characteristics can be tested in TAPPI bag biobleaching assay        (e.g., in triplicate) at a range of loadings and pH levels.

4. Exemplary Enzyme Characterization Screen—Temperature Profile

-   -   a. Thermotolerance of xylanases can be assayed using azo-xylan        assay at pH 8 and pH 10.4 at progressively more elevated        temperatures; and enzymes of the invention were tested using        this assay. The initial rates of reaction at each temperature        can be recorded and plotted to determine optimal performance        temperature of xylanases.    -   b. Residual activity—Another exemplary assay that can be        employed for testing thermostability of enzymes is the residual        activity method, whereby a sample of enzyme is treated at an        elevated temperature at a particular pH for a specific period of        time, and then assayed under standard conditions under        permissive temperature (typically 37° C.). A half-life at a        particular temperature is then determined and provides a measure        of a given enzyme fitness under those temperature conditions.

Example 8 Studies Demonstrating the Enzymatic Activity of Enzymes of theInvention

This example describes studies demonstrating the enzymatic activity ofthe exemplary xylanase enzymes of the invention, including the enzymaticactivity of any polypeptide of this invention, which includes apolypeptide having at 50% to 99% or more sequence identity to anexemplary enzyme of the invention, e.g., SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22 and/or SEQ ID NO:24, andalso includes any polypeptide having the sequence of SEQ ID NO:2 havingone or more amino acid residue changes (mutations) as set forth in Table1 and as described herein, have xylanase activity.

The evolution of endoxylanase SEQ ID NO:2 (Xyl 11) utilizing GSSMtechnology and xylanase screening identified point mutations (Xyl 11mutants) having increased xylanase activity, as well increased sugarrelease from alkaline pretreated corn stover, when used in combinationwith 7 other cellulosic enzymes (Table 2, below) after 36 hrs insaccharification cocktail assays at 50° C. These assays contain alkalinepretreated dry corn cobs at 5% (w/v) with a total enzyme loading of 10.2mg/g cellulose in the solids.

TABLE 2 Composition of enzyme cocktail Conc. mg/g Enzyme SEQ ID NOs:cellulose Endoglucanase^({circumflex over ( )}) SEQ ID NO: 4 1.7(encoded by SEQ ID NO: 3) Oligomerase I SEQ ID NO: 6 0.5 (beta-glucosidase)^({circumflex over ( )}) (encoded by SEQ ID NO: 5) CBH1 SEQID NO: 8 5 (GH family 7)^({circumflex over ( )}) (encoded by SEQ ID NO:7) CBH2 SEQ ID NO: 10 1 (GH family 6)^({circumflex over ( )}) (encodedby SEQ ID NO: 9) Xylanase VARIES 0.6 (GH family 11) (control*, Xyl 11 orXyl 11 mutants) Arabinofuranosidase^({circumflex over ( )}) SEQ ID NO:14 0.25 (encoded by SEQ ID NO: 13) Xylanase SEQ ID NO: 16 0.15 (GHfamily 10)^({circumflex over ( )}) (encoded by SEQ ID NO: 15)Oligomerase II SEQ ID NO: 18 1 (beta-xylosidase)^({circumflex over ( )}) (encoded by SEQ ID NO: 17) *controlxylanase is SEQ ID NO: 12 (encoded by SEQ ID NO: 11) {circumflex over( )}previously described in PCT Publication No. WO 07/094852

The new xylanase mutants improved xylose release over the wild type at0.6 mg/g cellulose as well as 0.2 mg/g cellulose loading (FIG. 2). Atthe standard loading of 0.6 mg/g cellulose these new variants achievedconversion rates of up to 90% monomeric xylose released vs. 63% with thewild type. Some of the polypeptides of the invention (the mutants of SEQID NO:2), in particular, Xyl 11 mutant 11 and Xyl 11 mutant 14, alsoachieved greater than 90% xylose release even at the reduced loading of0.2 mg cellulose. These novel polypeptides of the invention (the mutantsof SEQ ID NO:2) therefore not only improve the rate of xylose releasebut also can do so at a reduced enzyme loading. Similar positive effectson xylose release and enzyme loading could be envisioned for comparablesaccharification reactions using different feed stocks (switch grass,hard and soft woods, energy cane, bagasse etc.) applied to alkaline oracidic pretreatments and with different initial enzyme loadings (1mg-100 mg/g cellulose) and different ratios of cocktail components.

The enzymes of this invention can be used to process/treat cellulosicmaterial for, e.g., biological alcohol (e.g., EtOH, or ethanol)fermentation; cellulosic material that is processed using compositionsand methods of the invention can be mainly composed of cellulose(containing glucose), and hemicellulose-which is mostly containingxylose. In one aspect, glucose as well as xylose can be used as a sugarsource for EtOH fermentation. In one aspect, xylanases of the inventionare active in the enzymatic breakdown of the hemicellulose portion ofcellulosic material, releasing a monomeric xylose. In one aspect, theimproved xylanase activity of polypeptides of the invention increasesthe amount of xylose available for fermentation.

In one aspect, by removing the hemicellulose the cellulose becomes moreaccessible to cellulases, which can also increase the conversion ofcellulose to glucose. Using xylanases of the invention, e.g., thesequence variations of the exemplary endoxylanase Xyl 11 (SEQ ID NO:2),including the exemplary 18 amino acid substitutions described herein, anincreased specific activity can be achieved over the “wild type”xylanase, as described in Table 1, above. Note: in Table 1 tertiaryassay activity is indicated as the Absorbance at 560 nm measured in theBCA assay reached after 9.5 h of hydrolysis. Referencing Table 1, wheneach of these clones (xylanases of the invention) was evaluated incocktail saccharification assays with the xylanase as the variable,fourteen of these clones (xylanases of the invention) improved xyloseconversion rates when compared to assays with the wild type at the sameloading, as noted in Table 3 (see Table 1 for the sequence referenced inTable 3, e.g., Table 1 sets for the sequence of Xyl 11 mutant 5, Xyl 11mutant 5, etc., based on the exemplary SEQ ID NO:2; note also, “Xyl 11(WT)” refers to the “wild type” exemplary SEQ ID NO:2):

TABLE 3 Xylose conversion by the cocktail shown in Table 2 (above). Notethe xylanase component (Xyl 11 WT or Xyl 11 mutant) varies in eachcocktail. Xyl 11 or Xyl 11 mutant used in cocktail 36 hr STDEV XyloseConversion @ 0.2 mg Xyl 11 (WT) 51.57% 0.01 Xyl 11 mutant 5 55.98% 0.00Xyl 11 mutant 16 57.43% 0.01 Xyl 11 mutant 12 59.03% 0.02 Xyl 11 mutant4 59.46% 0.00 Xyl 11 mutant 9 60.34% 0.01 Xyl 11 mutant 17 60.45% 0.01Xyl 11 mutant 7 61.23% 0.02 Xyl 11 mutant 2 61.73% 0.02 Xyl 11 mutant 662.31% 0.00 Xyl 11 mutant 13 63.58% 0.01 Xyl 11 mutant 15 65.95% 0.00Xyl 11 mutant 10 66.34% 0.00 Xyl 11 mutant 11 71.76% 0.01 Xyl 11 mutant14 73.69% 0.03 Xylose Conversion @ 0.6 mg Xyl 11 (WT) 62.84% 0.02 Xyl 11mutant 16 68.10% 0.01 Xyl 11 mutant 5 69.69% 0.00 Xyl 11 mutant 1271.75% 0.01 Xyl 11 mutant 7 74.25% 0.00 Xyl 11 mutant 9 74.45% 0.00 Xyl11 mutant 17 74.76% 0.01 Xyl 11 mutant 4 74.94% 0.01 Xyl 11 mutant 1375.30% 0.01 Xyl 11 mutant 2 78.24% 0.05 Xyl 11 mutant 15 80.03% 0.00 Xyl11 mutant 6 80.36% 0.09 Xyl 11 mutant 10 80.61% 0.01 Xyl 11 mutant 1484.74% 0.01 Xyl 11 mutant 11 90.36% 0.06

Accordingly, the invention provides an enzyme cocktail comprising, orconsisting of, the enzymes: Endoglucanase, Oligomerase I(beta-glucosidase), CBH1 (GH family 7), CBH2 (GH family 6), Xylanase (GHfamily 11), Arabinofuranosidase, Xylanase (GH family 10) and anOligomerase II (beta-xylosidase); wherein one, two, three, four, five,six, seven and/or all eight of these enzyme are a polypeptide of thisinvention, and methods for treating polysaccharide compositions usingthese cocktails, or any cocktail of this invention, for, e.g.,treating/processing wood, pulp, paper, waste(s) and the like, or makingbiofuels or foods or feeds, or any other industrial process or method,e.g., as described herein.

Screens and Assays for Identifying Enzymes of the Invention

The following screens and assays were used in identifying exemplaryenzymes of the invention, and in one aspect, these screens and assayscan be applied to determine if any polypeptide has sufficient xylanaseactivity to fall with the scope of this invention—assuming of course italso has the requisite sequence identity, as described herein:

Xylanase evolution screen: Utilizing the GSSM technology (VereniumCorporation, U.S. Pat. No. 6,171,820) an evolution library forendoxylanase Xyl 11 (SEQ ID NO:2) representing all possible amino acidexchanges for each of the 194 residues of this enzyme was created. Pointmutations were introduced using degenerate oligonucleotides, one aminoacid position at a time, so that each original codon could besubstituted with each of the 20 naturally encoded amino acids. Themutated variants were transformed into XL1-Blue (recA-strain,Stratagene) and then into Pseudomonas fluorescens MB214 (Dow GlobalTechnologies Inc., US Patent Publication No. 20050130160), using vectorpWZ82T (SEQ ID NO:25). All variants were grown and expressed (fromPseudomonas fluorescens MB214) and lysed in 96 well plates. Hydrolysisreactions with the lysates were carried out in 96 well plates (200 ul of200 mM citrate buffer, pH 5.5, 0.5% dried and milled alkaline pretreatedcorn stover—CP-15, 50C). Aliquots were removed from the reaction at 1,3, 5 and 10 hrs and added to 800 mM carbonate buffer pH 10 to stop thereaction. The extent of hydrolysis at each time point was evaluated viaa reducing ends assay (BCA), as described by Johnston et al. 1998 (seebelow), recording absorption at 560 nm (A560). In addition aquantitative ELISA utilizing Xyl 11 (SEQ ID NO:2) specific antibodieswas used to normalize activity to protein expression. Both functionaland quantitative assays were automated for high through put. In theprimary screen, clones exhibiting normalized activity exceeding Xyl 11(SEQ ID NO:2) controls on the plate by at least 2 standard deviations(>1.0+2 STDV wt) were moved on to a secondary screen. In the secondaryscreen, all primary hits were re-screened in duplicate applying the sameassay and hit criteria as in the primary screen. Clones that confirmedfor both duplicates were then moved on to a tertiary screen. In tertiaryscreens, these clones again were assayed in duplicate using the BCAassay, but this time with different defined concentrations of protein(0.1, 0.05 and 0.025 mg/ml). Total protein of lysates was determined viaBradford assays (e.g., as described in Bradford 1976, see below) therelative content of xylanase then was determined via densitometry of SDSPAGE gels after running defined amounts of total protein. All clonesexceeding wt activity, recorded as absorption at 560 nm (A560), for atleast one enzyme concentration in the tertiary screen were then assayedin saccharification assays.

Saccharification/cocktail assay: Cocktail reactions were set up incapped 10 ml glass vials containing two metal ball bearings. Thereaction volume was 5 ml (200 mM Sodium Citrate-1 mM Sodium Azide pH5.5) with 5% solids (size 40 grit milled alkaline pretreated cornstover). Enzyme composition and loadings were according to Table 2,above, only varying the family 11 endoxylanase. Reaction vials wereincubated for 36 h at 50C under shaking at 300 rpm. The concentration ofxylose monomers released was determined by HPLC(R1 detector, ShodexSP-0810 column, flow rate of 0.5 ml/min) using a set of standards andcalibration curves.

-   -   Johnston, D. B.; Shoemaker, S. P.; Smith, G. M. and Whitaker, J.        R.: Kinetic Measurement of Cellulase Activity on Insoluble        Substrates Using Disodium 2,2′ Bicinchoninate. Journal of Food        Biochemistry (22) Issue 4 pp. 301-319, 1998    -   Bradford, M. M. (1976) A Rapid and Sensitive Method for the        Quantitation of Microgram Quantities of Protein Utilizing the        Principle of Protein-Dye Binding. Anal. Biochem. 72:248-25

While the invention has been described in detail with reference tocertain preferred aspects thereof, it will be understood thatmodifications and variations are within the spirit and scope of thatwhich is described and claimed.

1. An isolated, synthetic or recombinant nucleic acid comprising anucleic acid encoding a polypeptide having a xylanase activity, whereinthe nucleic acid comprises a sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity to SEQ ID NO:1 and has at least one mutation selected from (a)to (m), and wherein the said polypeptide shows an improved rate ofxylose release when compared to assays with SEQ ID NO: 2, at the sameenzyme loading, and shows an improved rate of xylose release whencompared to assays with SEQ ID NO: 2, at a reduced enzyme loading: (a)the nucleic acid codon encoding amino acid residue 38 of SEQ ID NO: 2 ischanged from CGT to CAC, and amino acid residue 38 Arginine, Arg (or“R”) is changed to Histidine, H is (or “H”); (b) the nucleic acid codonencoding amino acid residue 33 of SEQ ID NO: 2 is changed from CTG toGCG, and amino acid residue 33 Leucine, Leu (or “L”) is changed toAlanine, Ala (or “A”); (c) the nucleic acid codon encoding amino acidresidue 73 of SEQ ID NO: 2 is changed from GGC to TAC, and amino acidresidue 73 Glycine, Gly (or “G”) is changed to Tyrosine, “Tyr” (or “Y”);(d) the nucleic acid codon encoding amino acid residue 63 of SEQ ID NO:2 is changed from ATG to GTC, and amino acid residue 63 Isoleucine, Ile(or “I”) is changed to Valine, “Val” (or “V”); (e) the nucleic acidcodon encoding amino acid residue 188 of SEQ ID NO: 2 is changed fromAGC to GAG, and amino acid residue 188 Serine, Ser (or “S”) is changedto Glutamate, Glu (or “E”); (f) the nucleic acid codon encoding aminoacid residue 21 of SEQ ID NO: 2 is changed from TTC to TAC, and theamino acid residue 21 Phenylalanine, Phe (or “F”) is changed toTyrosine, “Tyr” (or “Y”); (g) the nucleic acid codon encoding amino acidresidue 125 of SEQ ID NO: 2 is changed from CAG to TAC, and amino acidresidue 125 Glutamine, Gln (or “Q”) is changed to Tyrosine, “Tyr” (or“Y”); (h) the nucleic acid codon encoding amino acid residue 4 of SEQ IDNO: 2 is changed from ACC to CGC, and amino acid residue 4 Threonine, or“Thr” (or “T”) is changed to Arginine, Arg (or “R”); (i) the nucleicacid codon encoding amino acid residue 73 of SEQ ID NO: 2 is changedfrom GGC to GAG, and amino acid residue 73 Glycine, Gly (or “G”) ischanged to Glutamate, Glu (or “E”); (j) the nucleic acid codon encodingamino acid residue 150 of SEQ ID NO: 2 is changed from GTA to GCC, andthe amino acid residue 150 Valine, Val (or “V”) is changed to Alanine,Ala (or “A”); (k) the nucleic acid codon encoding amino acid residue 17of SEQ ID NO: 2 is changed from TTC to GTC, and amino acid residue 17Phenylalanine, Phe (or “F”) is changed to Valine, “Val” (or “V”); (i)the nucleic acid codon encoding amino acid residue 4 of SEQ ID NO: 2 ischanged from ACC to CAC, and amino acid residue 4 Threonine, or “Thr”(or “T”) is changed to Histidine, H is (or “H”); and (m) the nucleicacid codon encoding amino acid residue 9 of SEQ ID NO: 2 is changed fromCCC to GAC, and the amino acid residue 9 Proline, Pro (or “P”) ischanged to Aspartate, Asp (or “D”);
 2. The isolated, synthetic orrecombinant nucleic acid comprising a nucleic acid encoding apolypeptide having a xylanase activity of claim 1, wherein the nucleicacid has one or more further nucleotide residue changes selected from:a) the nucleic acid codon encoding amino acid residue 4 of SEQ ID NO: 2is changed from ACC to AAC, and amino acid residue 4 is changed from Tto N; b) the nucleic acid codon encoding amino acid residue 4 of SEQ IDNO: 2 is changed from ACC to CGC, and amino acid residue 4 is changedfrom T to R; c) the nucleic acid codon encoding amino acid residue 4 ofSEQ ID NO: 2 is changed from ACC to CAC, and amino acid residue 4 ischanged from T to H; d) the nucleic acid codon encoding amino acidresidue 9 of SEQ ID NO: 2 is changed from CCC to GAC, and amino acidresidue 9 is changed from P to D; e) the nucleic acid codon encodingamino acid residue 17 of SEQ ID NO: 2 is changed from TTC to GTC, andamino acid residue 17 is changed from F to V; f) the nucleic acid codonencoding amino acid residue 21 of SEQ ID NO: 2 is changed from TTC toTAC, and amino acid residue 21 is changed from F to Y; g) the nucleicacid codon encoding amino acid residue 33 of SEQ ID NO: 2 is changedfrom CTG to GCG, and amino acid residue 33 is changed from L to A; h)the nucleic acid codon encoding amino acid residue 38 of SEQ ID NO: 2 ischanged from CGT to CAC, and amino acid residue 38 is changed from R toH; i) the nucleic acid codon encoding amino acid residue 44 of SEQ IDNO: 2 is changed from AGC to ACG, and amino acid residue 44 is changedfrom S to T; j) the nucleic acid codon encoding amino acid residue 63 ofSEQ ID NO: 2 is changed from ATC to GTC, and amino acid residue 63 ischanged from I to V; k) the nucleic acid codon encoding amino acidresidue 73 of SEQ ID NO: 2 is changed from GGC to TAC, and amino acidresidue 73 is changed from G to Y; l) the nucleic acid codon encodingamino acid residue 73 of SEQ ID NO: 2 is changed from GGC to GAG, andamino acid residue 73 is changed from G to E; m) the nucleic acid codonencoding amino acid residue 73 of SEQ ID NO: 2 is changed from GGC toGTC, and amino acid residue 73 is changed from G to V; n) the nucleicacid codon encoding amino acid residue 108 of SEQ ID NO: 2 is changedfrom TTC to AAG, and amino acid residue 108 is changed from F to K; o)the nucleic acid codon encoding amino acid residue 125 of SEQ ID NO: 2is changed from CAG to TAC, and amino acid residue 125 is changed from Qto Y; p) the nucleic acid codon encoding amino acid residue 150 of SEQID NO: 2 is changed from GTA to GCC, and amino acid residue 150 ischanged from V to A; q) the nucleic acid codon encoding amino acidresidue 188 of SEQ ID NO: 2 is changed from AGC to GAG, and amino acidresidue 188 is changed from S to E; and r) the nucleic acid codonencoding amino acid residue 189 of SEQ ID NO: 2 is changed from TCC toCAG, and amino acid residue 189 is changed from S to Q; wherein the saidpolypeptide shows an improved rate of xylose release when compared toassays with SEQ ID NO: 2, at the same enzyme loading, and shows animproved rate of xylose release when compared to assays with SEQ ID NO:2, at a reduced enzyme loading
 3. The nucleic acid of claim 1, whereinthe nucleic acid has one or more nucleic acid encodes at least onepolypeptide having at least one further amino acid residue change (orthe equivalent thereof) selected from: a) the amino acid residue 4 ischanged from T to N; b) the amino acid residue 4 is changed from T to R;c) the amino acid residue 4 is changed from T to H; d) the amino acidresidue 9 is changed from P to D; e) the amino acid residue 17 ischanged from F to V; f) the amino acid residue 21 is changed from F toY; g) the amino acid residue 33 is changed from L to A; h) the aminoacid residue 38 is changed from R to H; i) the amino acid residue 44 ischanged from S to T; j) the amino acid residue 63 is changed from I toV; k) the amino acid residue 73 is changed from G to Y; l) the aminoacid residue 73 is changed from G to E; m) the amino acid residue 73 ischanged from G to V; n) the amino acid residue 108 is changed from F toK; o) the amino acid residue 125 is changed from Q to Y; p) the aminoacid residue 150 is changed from V to A; q) the amino acid residue 188is changed from S to E; and r) the amino acid residue 189 is changedfrom S to Q; wherein the nucleic acid of claim 1 or 2 comprises asequence having at least 90% sequence identity to SEQ ID NO: 1 and itencodes a polypeptide which shows an improved rate of xylose releasewhen compared to assays with SEQ ID NO: 2, at the same enzyme loading,and shows an improved rate of xylose release when compared to assayswith SEQ ID NO: 2, at a reduced enzyme loading.
 4. The nucleic acid ofclaim 2, wherein the nucleic acid further comprises one or morenucleotide changes such that the nucleic acid encodes a polypeptidehaving at least one conservative amino acid substitution and retains itsxylanase activity; and, optionally, wherein the at least oneconservative amino acid substitution comprises substituting an aminoacid with another amino acid of like characteristics; or, a conservativesubstitution comprises a replacement of an aliphatic amino acid withanother aliphatic amino acid; replacement of a Serine with a Threonineor vice versa; replacement of an acidic residue with another acidicresidue; replacement of a residue bearing an amide group with anotherresidue bearing an amide group; exchange of a basic residue with anotherbasic residue; or replacement of an aromatic residue with anotheraromatic residue; and wherein the nucleic acid comprises a sequencehaving at least 85% sequence identity to SEQ ID NO: 1 and it encodes apolypeptide which shows an improved rate of xylose release when comparedto assays with SEQ ID NO: 2, at the same enzyme loading, and shows animproved rate of xylose release when compared to assays with SEQ ID NO:2, at a reduced enzyme loading.
 5. The nucleic acid of claim 2 whereincomprises a heterologous nucleic acid sequence or further comprises aheterologous nucleic acid sequence encoding a heterologous polypeptidesequence.
 6. The nucleic acid of claim 5, wherein the heterologouspolypeptide sequence comprises a heterologous signal sequence, aheterologous carbohydrate binding module (CBM), a heterologous dockerindomain, a heterologous catalytic domain (CD), or a combination thereof.7. The nucleic acid of claim 6, wherein the heterologous signalsequence, carbohydrate binding module or catalytic domain (CD) isderived from a heterologous enzyme; or a tag, an epitope, a targetingpeptide, a cleavable sequence, a detectable moiety or an enzyme.
 8. Thenucleic acid of claim 6, wherein the heterologous carbohydrate bindingmodule (CBM) comprises a xylan binding module, a cellulose bindingmodule, a lignin binding module, a xylose binding module, a mannansebinding module, a xyloglucan-specific module and/or anarabinofuranosidase binding module.
 9. The nucleic acid of claim 6, theheterologous signal sequence targets the encoded protein to a vacuole,the endoplasmic reticulum, a chloroplast or a starch granule.
 10. Anucleic acid sequence which is fully (completely) complementary to thesequence of claim
 1. 11. A vector comprising the sequence of claim 1,wherein the vector is a viral vector comprising an adenovirus vector, aretroviral vector or an adeno-associated viral vector.
 12. A transformedcell comprising a nucleic acid of claim 1 wherein the cell is abacterial cell, a mammalian cell, a fungal cell, a yeast cell, an insectcell or a plant cell.
 13. A transgenic plant, plant part or plant seed,or a transgenic non-human animal, comprising a nucleic acid of claim 1,wherein the plant is a corn plant, a sorghum plant, a potato plant, atomato plant, a wheat plant, an oilseed plant, a rapeseed plant, asoybean plant, a rice plant, a barley plant, a grass, a cotton plant, acottonseed plant, a palm, a sesame plant, a peanut plant, a sunflowerplant or a tobacco plant.
 14. An isolated, synthetic or recombinantpolypeptide or peptide having a xylanase activity, wherein thepolypeptide or peptide comprises a sequence having at least 95%, 96%,97%, 98%, 99%, or more, or complete (100%), sequence identity to SEQ IDNO:2 having at least one mutation selected from (a) to (m) and whereinthe said polypeptide with xylanase activity shows an improved rate ofxylose release when compared to assays with SEQ ID NO: 2, at the sameenzyme loading, and shows an improved rate of xylose release whencompared to assays with SEQ ID NO: 2, at a reduced enzyme loading: (a)the amino acid residue 38 Arginine, Arg (or “R”) is changed toHistidine, H is (or “H”)_(;) (b) the amino acid residue 33 Leucine, Leu(or “L”) is changed to Alanine, Ala (or “A”); (c) the amino acid residue73 Glycine, Gly (or “G”) is changed to Tyrosine, “Tyr” (or “Y”); (d) theamino acid residue 63 Isoleucine, Ile (or “I”) is changed to Valine,“Val” (or “V”); (e) the amino acid residue 188 Serine, Ser (or “S”) ischanged to Glutamate, Glu (or “E”); (f) the amino acid residue 21Phenylalanine, Phe (or “F”) is changed to Tyrosine, “Tyr” (or “Y”); (g)the amino acid residue 125 Glutamine, Gln (or “Q”) is changed toTyrosine, “Tyr” (or “Y”); (h) the amino acid residue 4 Threonine, or“Thr” (or “T”) is changed to Arginine, Arg (or “R”); (i) the amino acidresidue 73 Glycine, Gly (or “G”) is changed to Glutamate, Glu (or “E”);(j) the amino acid residue 150 Valine, Val (or “V”) is changed toAlanine, Ala (or “A”); (k) the amino acid residue 17 Phenylalanine, Phe(or “F”) is changed to Valine, “Val” (or “V”); (l) the amino acidresidue 4 Threonine, or “Thr” (or “T”) is changed to Histidine, H is (or“H”); and (m) the amino acid residue 9 Proline, Pro (or “P”) is changedto Aspartate, Asp (or “D”).
 15. The isolated, synthetic or recombinantpolypeptide or peptide of claim 14, wherein the polypeptide or peptidehas at least one of the following further amino acid residue changes: a)the amino acid residue 4 is changed from T to N; b) the amino acidresidue 4 is changed from T to R; c) the amino acid residue 4 is changedfrom T to H; d) the amino acid residue 9 is changed from P to D; e) theamino acid residue 17 is changed from F to V; f) the amino acid residue21 is changed from F to Y; g) the amino acid residue 33 is changed fromL to A; h) the amino acid residue 38 is changed from R to H; i) theamino acid residue 44 is changed from S to T; j) the amino acid residue63 is changed from I to V; k) the amino acid residue 73 is changed fromG to Y; l) the amino acid residue 73 is changed from G to E; m) theamino acid residue 73 is changed from G to V; n) the amino acid residue108 is changed from F to K; o) the amino acid residue 125 is changedfrom Q to Y; p) the amino acid residue 150 is changed from V to A; q)the amino acid residue 188 is changed from S to E; or r) the amino acidresidue 189 is changed from S to Q and wherein the polypeptide orpeptide comprises a sequence having at least 95% identity to SEQ ID NO:2 and shows an improved rate of xylose release when compared to assayswith SEQ ID NO: 2, at the same enzyme loading, and shows an improvedrate of xylose release when compared to assays with SEQ ID NO: 2, at areduced enzyme loading.
 16. The isolated, synthetic or recombinantpolypeptide or peptide of claim 14, wherein the polypeptide furthercomprises at least one conservative amino acid substitution and retainsits xylanase activity; wherein, optionally, the at least oneconservative amino acid substitution comprises substituting an aminoacid with another amino acid of like characteristics; or, a conservativesubstitution comprises: replacement of an aliphatic amino acid withanother aliphatic amino acid; replacement of a Serine with a Threonineor vice versa; replacement of an acidic residue with another acidicresidue; replacement of a residue bearing an amide group with anotherresidue bearing an amide group; exchange of a basic residue with anotherbasic residue; or replacement of an aromatic residue with anotheraromatic residue; wherein the polypeptide or peptide comprises asequence having at least 95% identity to SEQ ID NO: 2 and shows animproved rate of xylose release when compared to assays with SEQ ID NO:2, at the same enzyme loading, and shows an improved rate of xyloserelease when compared to assays with SEQ ID NO: 2, at a reduced enzymeloading.
 17. The isolated, synthetic or recombinant polypeptide orpeptide of claim 14, wherein the polypeptide or peptide is encoded bythe nucleic acid of claim
 1. 18. The isolated, synthetic or recombinantpolypeptide or peptide of claim 14, wherein the polypeptide or peptidefurther comprises a heterologous amino or polypeptide or peptidesequence.
 19. The isolated, synthetic or recombinant polypeptide orpeptide of claim 18, wherein the heterologous amino or polypeptide orpeptide sequence comprises, a heterologous signal sequence, aheterologous carbohydrate binding module (CBM), a heterologous dockerindomain, a heterologous catalytic domain (CD), a tag, an epitope, atargeting peptide, a cleavable sequence, a detectable moiety or anenzyme, or a combination thereof.
 20. The isolated, synthetic orrecombinant polypeptide or peptide of claim 19, wherein the heterologoussignal sequence, carbohydrate binding module or catalytic domain (CD) isderived from a heterologous enzyme.
 21. The isolated, synthetic orrecombinant polypeptide or peptide of claim 19, wherein the heterologouscarbohydrate binding module (CBM) comprises a xylan binding module, acellulose binding module, a lignin binding module, a xylose bindingmodule, a mannanse binding module, a xyloglucan-specific module and/oran arabinofuranosidase binding module, or a combination thereof.
 22. Theisolated, synthetic or recombinant polypeptide or peptide of claim 19,wherein the heterologous signal sequence targets the encoded protein toa vacuole, the endoplasmic reticulum, a chloroplast or a starch granule.23. A protein preparation, or an immobilized polypeptide, comprising thepolypeptide of claim 14, wherein the protein preparation comprises aliquid, a solid or a gel.
 24. An isolated, synthetic or recombinantantibody that specifically binds to the polypeptide of claim 14, whereinoptionally the antibody is a monoclonal or a polyclonal antibody, or isa single chained antibody.
 25. An industrial process or a method forusing a polypeptide having a xylanase activity, wherein the polypeptidecomprises an amino acid sequence of claim 14, or the polypeptide isencoded by a nucleic acid of claim 1, or enzymatically active fragmentsthereof; comprising: (a) a method for hydrolyzing, liquefying, breakingup or disrupting a xylan-, cellulose- or hemicellulose-comprisingcomposition comprising contacting the polypeptide or enzymaticallyactive fragments thereof with a composition comprising a xylan, acellulose or a hemicellulose under conditions wherein the polypeptide orenzymatically active fragments thereof hydrolyze, liquefy, break up ordisrupt the xylan-, cellulose- or hemicellulose-comprising composition,wherein optionally the composition comprises a plant cell or a bacterialcell, (b) a method for reducing the amount of lignin (delignification),or solubilizing a lignin, in a paper or paper product, a wood, wood pulpor wood product, or a wood or paper recycling composition, comprisingcontacting the paper or paper product, wood, wood pulp or wood product,or wood or paper recycling composition with the polypeptide orenzymatically active fragments thereof; (c) a method for hydrolyzingcelluloses, hemicelluloses or xylans in a biomass, a wood, wood product,paper pulp, paper product or paper waste comprising contacting the wood,wood product, paper pulp, paper product or paper waste with thepolypeptide or enzymatically active fragments thereof; (d) a method forenzymatic decoloring of paper, hemp or flax pulp comprising contactingthe paper, hemp or flax pulp with the polypeptide or enzymaticallyactive fragments thereof and a bleaching agent, wherein optionally thedecoloring agent comprises oxygen or hydrogen peroxide under conditionssuitable for enzymatic decoloring; (e) a method for of decoloring alignocellulose pulp comprising contacting the lignocellulose pulp withthe polypeptide or enzymatically active fragments thereof underconditions suitable for enzymatic decoloring; (f) a method for enzymaticdeinking of paper, paper waste, paper recycled product, deinking tonerfrom non-contact printed wastepaper or mixtures of non-contact andcontact printed wastepaper, comprising contacting the paper, paperwaste, paper recycled product, non-contact printed wastepaper or contactprinted wastepaper with the polypeptide or enzymatically activefragments thereof, under conditions suitable for enzymatic deinking; (g)a method for decoloring or deinking newspaper comprising contacting thenewspaper with the polypeptide or enzymatically active fragments thereofunder conditions suitable for enzymatic decoloring or deinking; or (h)method for reducing lignin in a composition selected from a wood or woodproduct, a wood pulp, a Kraft pulp, a paper, a paper product or a paperpulp, comprising contacting the composition with the polypeptide orenzymatically active fragments thereof under conditions suitable forenzymatic reducing of the lignin.
 26. An enzyme mixture or cocktailcomprising: (a) at least one polypeptide of claim 14, or a polypeptideencoded by a nucleic acid of claim 1, and one or more other enzyme(s);or (b) the mixture or cocktail of (a), wherein the one or more otherenzyme(s) is another xylanase, a mannanase and/or a glucanase,cellulases, lipases, esterases, proteases, or endoglycosidases,endo-beta-1,4-glucanases, beta-glucanases, endo-beta-1,3(4)-glucanases,cutinases, peroxidases, catalases, laccases, amylases, glucoamylases,pectinases, reductases, oxidases, phenoloxidases, ligninases,pullulanases, arabinanases, hemicellulases, mannanases, xyloglucanases,xylanase, a mannanase and/or a glucanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases.
 27. Aprocess for hydrolyzing xylans, celluloses or hemicelluloses in anyorganic compound, plant or wood or wood product or byproduct, woodwaste, paper pulp, paper product or paper waste or byproduct comprisinguse of a polypeptide of claim 14, or a polypeptide encoded by a nucleicacid of claim 1, or an enzymatically active fragment thereof, or theenzyme mixture or cocktail of claim 26, or a combination thereof.
 28. Aliquid composition comprising: (a) (i) an alcohol and (ii) a polypeptideof claim 14, or a polypeptide encoded by a nucleic acid of claim 1, oran enzymatically active fragment thereof, or the enzyme mixture orcocktail of claim 26, or a combination thereof; (b) the liquidcomposition of (a), wherein the alcohol is or comprises ethanol,propanol, butanol and/or methanol; or (c) the liquid composition of (a)or (b) comprising or contained in a fuels, a biofuel, a synthetic liquidor gas or a syngas.
 29. The enzyme mixture or cocktail of claim 26,comprising an Endo glucanase, an Oligomerase I (beta-glucosidase), aCBH1 (GH family 7), a CBH2 (GH family 6), a Xylanase (GH family 11), anArabinofuranosidase, a Xylanase (GH family 10) and an Oligomerase II(beta-xylosidase), wherein at least one of these enzymes is apolypeptide of claim 14, or a polypeptide encoded by a nucleic acid ofclaim 1.